KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Rabbit polyclonalanti-PAFAcetylhydrolase (human) antibody | Cayman Chemical | Cat#160603; Clone#N/A; RRID:AB_10077761. |
| Rabbit polyclonalanti-PAFAcetylhydrolase (human) antibody (used for IP) | SHANGHAI GENOMICS,INC. | Cat# N/A; Clone# N/A; RRID:N/A |
| Rabbit monoclonal anti-AKT1 antibody | Cell Signaling Technology | Cat#75692; Clone#D9R8K; RRID: AB_2716309. |
| Rabbit monoclonal anti-P-AKT (S473) antibody | Cell Signaling Technology | Cat#4060S; Clone#D9E; RRID: AB_2315049. |
| Rabbit monoclonal anti-P-MEK1/2 (S217/221) antibody | Cell Signaling Technology | Cat# 9154S; Clone# 41G9; RRID: AB_2138017. |
| Rabbit monoclonal anti-MEK1/2 antibody | Cell Signaling Technology | Cat# 8727S; Clone# D1A5; RRID: AB_10829473. |
| Rabbit polyclonal anti-p44/42 MAP kinase (phosphorylated ERK1/2) antibody | Cell Signaling Technology | Cat# 9101L; Clone# N/A; RRID: AB_331646. |
| Rabbit polyclonal anti-ERK1/2 antibody | Cell Signaling Technology | Cat# 9102S; Clone# N/A; RRID: AB_330744. |
| Mouse monoclonal anti-β-actin antibody | Sigma-Aldrich | Cat# A1978; Clone# AC-15; RRID: AB_476692. |
| Rabbit monoclonal anti-BRAFantibody | Cell Signaling Technology | Cat# 14814S; Clone# D9T6S; RRID:AB_2750887. |
| Rabbit monoclonal anti-RAF1 antibody | Cell Signaling Technology | Cat# 53745S; Clone# D4B3J; RRID:AB_2799444. |
| Rabbit polyclonal anti-Phospho-RAF1(Ser259) antibody | Cell Signaling Technology | Cat# 9421S; Clone# N/A; RRID: AB_330759. |
| Rabbit monoclonalanti-Phospho-RAF1 (Ser338) antibody | Cell Signaling Technology | Cat# 9427S; Clone# 56A6; RRID:AB_2067317. |
| Rabbit polyclonal anti-PAK1 antibody | Cell Signaling Technology | Cat# 2602S; Clone#N/A; RRID: AB_330222. |
| Rabbit polyclonal anti-PAK2 antibody | Cell Signaling Technology | Cat# 2608S; Clone# N/A; RRID:AB_2283388. |
| Rabbit polyclonal anti-PAK3 antibody | Cell Signaling Technology | Cat# 2609S; Clone#N/A; RRID:AB_2225298. |
| Mouse monoclonal anti-rabbit IgG (Conformation Specific) (HRP Conjugate) antibody | Cell Signaling Technology | Cat# 5127; Clone# L27A9; RRID:AB_10892860. |
| Rabbit polyclonal anti-Phospho-PAK1 (Thr423)/PAK2 (Thr402)antibody | Cell Signaling Technology | Cat# 2601; Clone# N/A; RRID:AB_330220. |
| Rabbit polyclonal anti-Phospho-PAK1 (Ser144)/PAK2 (Ser141) antibody | Cell Signaling Technology | Cat# 2606S; Clone# N/A; RRID:AB_2299279. |
| Rabbit polyclonal anti-Phospho-PAK1 (Ser199/204)/PAK2 (Ser192/197) antibody | Cell Signaling Technology | Cat# 2605S; Clone# N/A; RRID:AB_2160222. |
| Rabbit monoclonal anti-FLAG antibody | Cell Signaling Technology | Cat# 14793S; Clone# N/A; RRID: AB_2572291. |
| anti-FLAG(R) M2 Affinity Gel antibody | Sigma- Aldrich | Cat# A2220-5ML; Clone#N/A; RRID: AB_10063035. |
| Rabbit polyclonal Anti-Phospho-(Ser/Thr) Phe antibody | Abcam | Cat# ab17464; Clone# N/A; RRID: AB_443891. |
| Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP | Thermo Fisher Scientific | Cat# 31430; Clone# N/A; RRID: AB_228307 |
| Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP | Thermo Fisher Scientific | Cat# 31460; Clone# N/A; RRID: AB_ 228341 |
| Rabbit monoclonal anti-CDC42 antibody | Cell Signaling Technology | Cat# 2466S; Clone# 11A11; RRID: AB_2078082. |
| Rabbit polyclonal anti-alpha-Tubulin antibody | Cell Signaling Technology | Cat# 2144S; Clone# N/A; RRID: AB_2210548 |
| Rabbit monoclonal anti-GAPDH antibody | Cell Signaling Technology | Cat# 2118S; Clone# 14C10; RRID: AB_561053 |
| Bacterial and Virus Strains | ||
| BL21(DE3) Chemically Competent Cells | Sigma-Aldrich | Cat# CMC0014 |
| DH10B Competent Cells | Thermo Fisher Scientific | Cat#EC0113 |
| One Shot™ ccdB Survival Cells | Thermo Fisher Scientific | Cat#11828029 |
| Biological Samples | ||
| N/A | ||
| Chemicals, Peptides, and Recombinant Proteins | ||
| Platelet-activating factor (PAF) | AVANTI | Cat# 840009C CAS: 65154-06-5 |
| Lyso-PAF | AVANTI | Cat# 840008C CAS: 52691-62-0 |
| D-Sphingosine | Sigma-Aldrich | Cat#S7049-CAS: 123-78-4 |
| Trypan Blue | Sigma-Aldrich | Cat# T8154-100ML |
| Adenosine 5’-triphosphate (ATP) disodium salt hydrate | Sigma-Aldrich | Cat# A1852 CAS: 34369-07-8 |
| POLYBRENE | American Bioanalytical | Cat#AB01643-00001 |
| Puromycin dihydrochloride | Sigma-Aldrich | Cat# P8833 CAS: 58-58-2 |
| Hygromycin B | emdmillipore | Cat# 400052 CAS: 31282-04-9 |
| [3H]-PAF | ARC | Cat#ART 0727-50 μ;Ci |
| [3H]-Lyso-PAF | ARC | Cat#ART 1741-250 uCi |
| [α-32P]-ATP | PerkinElmer | Cat#BLU003X250UC |
| Recombinant human PAK1 protein | Abcam | Cat#ab125805 |
| Recombinant human PLA2G7 protein | Sigma-Aldrich | Cat#SRP3136 |
| Myelin Basic Protein bovine | Sigma-Aldrich | Cat# M1891 |
| COMPLETE, EDTA-FREE, 20 TABLETS | Sigma-Aldrich | Cat#11873580001 |
| EDTA-free protease and phosphatase inhibitor Cocktail | Thermo Fisher Scientific | Cat# A32961 |
| L-GLUTATHIONE REDUCED | Sigma-Aldrich | Cat#G4251-10G |
| Critical Commercial Assays | ||
| TransIT®-LT1 Transfection Reagent | Mirus | Cat# MIR 2305 |
| PAF Acetylhydrolase Assay Kit | Cayman Chemical | Cat#760901 |
| Human PLA2G7 SimpleStep ELISAKit | Abcam | Cat#ab235643 |
| Lipofectamine® 3000 | Life Technologies | Cat#L3000015 |
| Lipofectamine® RNAiMAX Transfection Reagent | Life Technologies | Cat#13778030 |
| Phusion® High-Fidelity DNA Polymerase | New England Biolabs | Cat#M0530S |
| QIAquick Gel Extraction Kit | Qiagen | Cat# 28704 |
| QIAprep Spin Miniprep Kit | Qiagen | Cat#27106 |
| pENTER/D-TOPO CLONING KIT | Invitrogen | Cat#45-021-8 |
| Gateway™ LR Clonase™ II Enzyme Mix | Invitrogen | Cat#11791020 |
| AccuPrime™ Pfx DNA Polymerase | Invitrogen | Cat#12-344-024 |
| Protein G Sepharose® 4 Fast Flow | Sigma-Aldrich | Cat# GE17-0618-01 |
| Glutathione Sepharose® 4B | GE Healthcare, | Cat#17-0756-01 |
| ADP-Glo™ Kinase Assay Kit | Promega | Cat#V9101 |
| ProBond™ Resin | Life Technologies | Cat#460019 |
| Disposable PD-10 Desalting Columns | GE Healthcare | Cat#95017-001 (PK) |
| Pierce™ Rapid Gold BCA Protein Assay Kit | Thermo Fisher Scientific | Cat#A53225 |
| Coomassie Brilliant Blue R-250 Staining Solution | Bio-rad | Cat# 1610436 |
| Deposited Data | ||
| Original images were deposited to Mendeley data | This paper | https://data.mendeley.com/datasets/g6nfy3hnzf/draft?a=40ef7051-f2e5-4658-8f5b-ef7e008dcd3f |
| Experimental Models: Cell Lines | ||
| Human: HEK293T cells | ATCC | Cat# CRL-3216; RRID:CVCL_0063 |
| Human: A375 cells | ATCC | Cat# CRL-1619; RRID: CVCL_0132 |
| Human: A2058 cells | ATCC | Cat# CRL-11147; RRID: CVCL_1059 |
| Human: SK-MEL-5 cells | ATCC | Cat# HTB-70; RRID: CVCL_0527 |
| Human: WM266-4 cells | ROCKLAND IMMUNOCHEMICALS | Cat#WM266-4-01-0001; RRID: N/A |
| Human: SK-MEL-2 cells | ATCC | Cat# HTB-68; RRID: CVCL_0069 |
| Human: HMCB cells | ATCC | Cat# CRL-9607; RRID: CVCL_3317 |
| Human:VMM39 | ATCC | Cat# CRL-3230; RRID: CVCL_A739 |
| Human:VMM1cells | ATCC | Cat# CRL-3225; RRID: CVCL_A732 |
| Human:NRAS mutant-A375 Isogenic Cell Line | ATCC | Cat# CRL-1619IG-2; RRID: CVCL_JF22 |
| Human: PMWK cells | ATCC | Cat# CRL-2624; RRID: CVCL_A665 |
| Human: CHL-1 cells | ATCC | Cat# CRL-9446; RRID: CVCL_1122 |
| Human: Mel-ST cells | Kang et al., 2015 (KHB MC paper) | Cat# N/A; RRID: N/A |
| Human: WM1985 cells | ROCKLAND IMMUNOCHEMICALS | Cat# WM1985-01-0001; RRID: N/A |
| Human: WM164 cells | ROCKLAND IMMUNOCHEMICALS | Cat# WM164-01-0001; RRID: N/A |
| Human: WM2032 cells | ROCKLAND IMMUNOCHEMICALS | Cat# WM2032-01-0001; RRID: N/A |
| Human: HCT116 cells | ATCC | Cat# CCL-247; RRID:CVCL_0291 |
| Human: HT-29 cells | ATCC | Cat# HTB-38; RRID: CVCL_0320 |
| Experimental Models: Organisms/Strains | ||
| Mouse: Heterozygous Hsd:Athymic Nude-Foxn1nu/Foxn1+ | Envigo/Harlan | Cat# 069(nu)/070(nu/+) |
| Oligonucleotides | ||
| shRNA targeting PLA2G7 sequence #3: 5’-TCAGGACACTTTATTCTGCTA -3’ | This paper | N/A |
| shRNA targeting PLA2G7 sequence #4: 5’-CACCCTTTGGATCCCAAATAA -3’ | This paper | N/A |
| Control siRNA sequence: 5’-UUCUCCGAACGUGUCACGUdTdT-3’ | Qiagen | Cat# 1022076 |
| siRNA targeting PLA2G7 sequence #3: 5’- TCAGGACACTTTATTCTGCTA -3’ | Qiagen | Cat# SI00072191 |
| siRNA targeting PLA2G7 sequence #4: 5’- CACCCTTTGGATCCCAAATAA -3’ | Qiagen | Cat# SI00072198 |
| shRNA targeting BRAF sequence: 5’-GCTGGTTTCCAAACAGAGGAT-3’ | BROAD Institute/Open Biosystems | N/A |
| siRNA targeting PAK1 sequence: 5’-CAUCAAAUAUCACUAAGUC-3’ | Dharmacon | Cat# D-003521-03-0002 |
| siRNA targeting PAK2 sequence: 5’-AGAAGGAACUGAUCAUUAA-3’ | Dharmacon | Cat# D-003597-05-0002 |
| siRNA targeting CDC42 | Sigma-Aldrich | Cat#EHU117241 |
| Primer: PLA2G7, Forward: 5’-CACCATGGTGCCACCCAAATTGCATG-3’ | This paper | N/A |
| Primer: PLA2G7, Reverse:5’-TTACTTGTCATCGTCGTCCTTGTAGTCATTGTATTTCTCTATTCCTG-3’ | This paper | N/A |
| Primers: PLA2G7 S273A mutation, Forward: 5’-GCAGTAATTGGACATGCTTTTGGTGGAGCAAC-3’ | This paper | N/A |
| Primers: PLA2G7 S273A mutation, Reverse: 5’-GTTGCTCCACCAAAAGCATGTCCAATTACTGC -3’ | This paper | N/A |
| Primers: PAK2, Forward: 5’-CACCATGTCTGATAACGGAGAAC -3’ | This paper | N/A |
| Primers:PAK2, Reverse: 5’-TTACTTGTCATCGTCGTCCTTGTAGTCACGGTTACTCTTCATTGCTT -3’ | This paper | N/A |
| Primer: PAK2 Catalytic domain, Forward: 5’-CACCATGGAAGAGATTATGGAGAAATT -3’ | This paper | N/A |
| Recombinant DNA | ||
| pLKO.1 puro-shPLA2G7#3 | This paper | N/A |
| pLKO.1 puro-shPLA2G7#4 | This paper | N/A |
| LentiCRISPRv2-sgPLA2G7 | GenScript | Cat# SC1678 |
| pLKO.1 puro-shBRAF | This paper | N/A |
| pLKO.1 hygro-shPLA2G7#3 | This paper | N/A |
| pDEST27-FLAG-PLA2G7 (RNAi-resistant) | This paper | N/A |
| pDEST27-FLAG-PLA2G7-S273A (RNAi-resistant) | This paper | N/A |
| pLKO.1 hygro-FLAG-PLA2G7 | This paper | N/A |
| pDEST27-MEK-DD | Kang et al., 2015 (KHB MC paper) | N/A |
| pDEST27-BRAF-V600E | Kang et al., 2015 (KHB MC paper) | N/A |
| pET60-FLAG-PAK2 | This paper | N/A |
| pET60-FLAG-PAK2 Catalytic domain | This paper | N/A |
| pET53-FLAG-PAK2 Catalytic domain | This paper | N/A |
| pDONR223-PAK2 | pDONR223-PAK2 was a gift from William Hahn & David Root. | Addgene plasmid: # 23655; RRID:Addgene_23655 |
| pDONR223-RAF1 | pDONR223-RAF1 was a gift from William Hahn & David Root. | Addgene plasmid: # 23832; RRID:Addgene_23832 |
| pET60-RAF1 | This paper | N/A |
| pET53-MEK | Kang et al., 2015 (KHB MC paper) | N/A |
| Software and Algorithms | ||
| Human pLKO.1 the RNAi consortium (TRC) Library | BROAD Institute/ Open Biosystems | https://www.broadinstitute.org/rnai-consortium/rnai-consortium-shrna-library |
| GraphPad Prism 5 software | GraphPad Software | https://www.graphpad.com/; RRID:SCR_002798 |
| ImageJ | https://imagej.nih.gov/ij/ | RRID:SCR_003070 |