. 2020 Oct 13;9:e57117. doi: 10.7554/eLife.57117

Table 1. HELLS is co-immunoprecipitated with PRDM9.

Two independent immunoprecipitation experiments were performed using HeLa cells and mouse testis extracts. In the first experiment, HeLa S3 cells that express N-terminally (Nter) or C-terminally (Cter) tagged human PRDM9 or without PRDM9 expression vector (no PRDM9) were used to identify proteins that interacts with PRDM9 after size selection (95–120 kD and 70–80 kD), and without size selection. Mouse testis extracts were prepared without (rep1) or after incubation with benzonase (rep2) (in duplicate). IP were performed with an anti-PRDM9 antibody or with normal rabbit serum (mock). For each protein (PRDM9 and HELLS), the total number of peptides, the protein rank in the whole set of proteins with at least one peptide, and ranked by number of peptides, and the sequence coverage are indicated; na: not applicable. For mouse testis extracts, the rank difference of the label free quantification intensity (LFQ) between IPs with anti-PRDM9 and mock are indicated. The full list of the identified peptides is in Supplementary file 1. Extracts analysis by electrophoresis are presented in Table 1—source data 1.

Table 1—source data 1. Purification of protein complexes.

(A) Western blot analysis after complex purification by Flag-HA of extracts from HeLa S3 cells. HeLa S3 cells without PRDM9 expression vector, or expressing human PRDM9 tagged with Flag-HA at the C-terminus (PRDM9-Ct) or N-terminus (PRDM9-Nt) were used. Protein fractions of the extracts before IP (S1: cytoplasmic fraction, S2: nuclear fraction as input for IPs, ppt: insoluble pellet) and after the affinity purification steps were analyzed by western blotting using an anti-PRDM9 antibody. (B) Analysis of affinity-purified proteins after silver staining (sample without size selection). Eluates 1, 2 and resin fractions obtained from affinity purification (HA) of extracts initially prepared from HeLa S3 cells without PRDM9 expression vector (M), or expressing human PRDM9 tagged with Flag-HA at the C-terminus (Ct) or at the N-terminus (Nt) were separated by electrophoresis and silver stained. Mixtures of Eluate 1 and 2 were used for mass spectrometry analysis. (C) Western blot analysis of complex purification using an anti-PRDM9 antibody and mouse testes extracts (Mouse testis rep1). Protein extracts obtained during the Dignam-based purification (S1: cytoplasmic fraction, S2: nuclear fraction, S3: DNase-treated, and ppt: pellet) were loaded. Input (S2), unbound (UB), and proteins immunoprecipitated (IP) with an anti-PRDM9 antibody or normal rabbit serum (mock) were analyzed by western blotting. Detection was with an anti-PRDM9 antibody. Loading: 1 and 10% of input and IP samples, respectively. (D) Analysis of affinity-purified proteins by silver staining (Mouse testis rep1). Input, and samples IP with an anti-PRDM9 antibody or with normal rabbit serum (mock) were loaded. Bovine serum albumin (BSA) was used as control. Proteins were separated by electrophoresis and silver stained. (E) Western blot analysis of complex purification using an anti-PRDM9 antibody in extracts from mouse testes incubated with benzonase (Mouse testis rep 2), in duplicate (a and b). Protein extracts obtained during the Dignam-based purification steps (S1: cytoplasmic fraction, S2: nuclear fraction, S3: DNase-treated, and ppt: pellet) were loaded. Input (S2) and proteins IP with an anti-PRDM9 antibody or rabbit serum (mock) were analyzed by western blotting. Detection was with an anti-PRDM9 antibody. Loading: 1% and 10% of input and IP samples. (F) Analysis of affinity purified proteins by silver staining (Mouse testis rep2). Input, and samples IP with an anti-PRDM9 antibody or with normal rabbit serum (mock) were loaded. BSA was used as control. Proteins were separated by electrophoresis and stained with silver.

elife-57117-table1-data1.pdf^{(1.2MB, pdf)}

IP	Total peptides PRDM9	Rank PRDM9	Sequence coverage PRDM9 (%)	Total peptides HELLS	Rank HELLS	Sequence coverage HELLS (%)
HeLa with size selection
HeLa PRDM9 Nter 95–120 KD 70–80 KD	0 7	na 1/21	na 6.4	11 0	1/16 na	11.9 na
HeLa PRDM9 Cter 95–120 KD 70–80 KD	0 7	na 3/34	na 7.3	4 0	2/32 na	4.4 na
HeLa no PRDM9 95–120 KD 70–80 KD	0 0	na	na	0 0	na	na
HeLa without size selection
HeLa PRDM9 Nter	38	6/447	29.4	97	1/447	48.1
HeLa PRDM9 Cter	35	4/364	33.7	44	1/364	37.6
HeLa no PRDM9	0	na	na	0	na	na
Mouse testis rep1
IP PRDM9	24	27/571	35.1	14	75/571	24.1
mock	1	538/571	1.2	6	211/571	9.3
LFQ Rank difference		441			113
Mouse testis rep2 (+benzonase)
IP PRDM9	14 15	39/890 41/948	26.3	6 7	187/890 178/948	11
mock	1 0	782/890 na	1.4	5 1	323/890 506/948	7.2
LFQ Rank difference		870 688			122 468