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. Author manuscript; available in PMC: 2015 Mar 1.
Published in final edited form as: FEBS J. 2014 Feb 13;281(6):1571–1584. doi: 10.1111/febs.12724

Figure 4. SSTY proteins interact with SLX/SLY proteins.

Figure 4

(A) Anti-SSTY1 antibody detection of protein extracts from COS-7 cells transfected with SSTY1MYC and either FLAGSLY1, FLAGSLY2, FLAGSLX, FLAGSLXL1 or FLAGSLYCterm, before (INPUT) and after immunoprecipitation (IP) with FLAG antibody. SSTY1MYC (indicated by an arrow) is immunoprecipitated by FLAG antibody when co-expressed with FLAGSLY1, FLAGSLY2, FLAGSLX, FLAGSLXL1 but not with FLAGSLYCterm. (B) Anti-MYC antibody and anti-SSTY1/2 antibody detection of protein extracts from COS-7 cells transfected with SSTY2MYC and either FLAGSLX, FLAGSLY1, FLAGFLAGSLY2, FLAGSLXL1, FLAGSLY37AA or FLAGSLYCterm, before (INPUT) and after immunoprecipitation (IP) with FLAG antibody. The star indicates the band corresponding to the light chain of the antibody used for immunoprecipitation. SSTY2MYC (indicated by an arrow) is immunoprecipitated by FLAG antibody when co-expressed with FLAGSLY1, FLAGSLY2, FLAGSLX, FLAGSLXL1, FLAGSLY37AA but not with FLAGSLYCterm. (C) Anti-FLAG detection of protein extracts from COS-7 cells transfected with SSTY1MYC and either FLAGSLY1, FLAGSLY1Nterm, FLAGSLY37AA or FLAGSLYCterm, before (INPUT) and after immunoprecipitation (IP) with MYC antibody. On the right are indicated the bands of the protein molecular weight ladder (35KDa, 25kDa and 15KDa). Anti-FLAG detection shows the presence of FLAGSLY1 protein (~38kDa), FLAGSLY1Nterm (~15kDa), FLAGSLY37AA (~15kDa) and FLAGSLYCterm (~10kDa) peptides (from left to right) before immunoprecipitation (INPUT). Anti-FLAG detection of the immunoprecipitated samples (MYC IP) shows that FLAGSLY1, FLAGSLY1Nterm and FLAGSLY37AA were immunoprecipitated by MYC antibody but not FLAGSLYCterm. In the lane corresponding to FLAGSLY1 immunoprecipitated by MYC antibody, four bands are detected with anti-FLAG antibody. The ~38kDa band corresponds to the intact version of FLAGSLY1, the ~28kDa, ~18kDa and ~15kDa bands likely correspond to shorter version of FLAGSLY1 protein which were produced by proteolysis during the immunoprecipitation process. (D) SSTY1 antibody detection of wild type (WT) and Sly-deficient (shSLY) whole testicular extracts immunoprecipitated either with anti-SLX/SLXL1 antibody or with SLX/SLXL1 pre-immune serum (negative control). SLX/SLXL1 interacts in vivo with SSTY1 (E) CLUSTALW alignment of SLX (GenBank accession number NP_001129948.1), SLXL1 (NP_083457.1), SLY1 (NP_963288.2) and SLY2 (NP_001032837.2) protein sequences. Stars indicate identities and dots similarities (one or two dots indicate strong or weaker conservative changes, respectively), a gap indicates no conservation. The sequence corresponding to SLY1Nterm is underlined once; the sequence corresponding to SLY37AA construct (the minimal domain of interaction with SSTY), which overlaps with part of SLY1Nterm, is underlined twice; the Cor1 domain (as defined by NCBI Conserved Domain), representing SLY1Cterm, is highlighted in grey.