control plasmid with pspax2  (Addgene inc)

Journal: EMBO Reports

Article Title: Phosphatase PTPN22 functions as an adaptor in the mTORC2 complex

doi: 10.1038/s44319-025-00576-5

Figure Lengend Snippet: ( A ) A schematic showing sensitivity of mTORC2 towards different detergents during isolation of mTORC2. Cell lysis with 0.3% CHAPS buffer preserves mTORC2 integrity, while 1% Triton X-100 containing buffer cause dissociation of mTORC2 into two modules; one having mTOR and mLST8, and other having RICTOR and mSIN. ( B ) Immunoprecipitation (IP) with control IgG or anti-PTPN22 antibody was performed with extracts derived from Jurkat cells lysed in a buffer containing either 0.3% CHAPS or 1% Triton X-100. Endogenous association of PTPN22 with mTORC2 components were analysed by immunoblotting with respective antibodies. ( C ) Bacterially expressed recombinant MBP or MBP-PTPN22 proteins immobilized to dextran Sepharose beads were incubated with concentrated bacterial cell lysates expressing GST-RICTOR. The MBP-pulldowns were resolved by SDS-PAGE and the interactions were analyzed by immunoblotting with anti-RICTOR antibody. Expression of MBP and MBP-PTPN22 was shown by Coomassie staining. ( D ) Bacterially expressed recombinant GST, GST-mSIN and MBP-PTPN22 were purified using glutathione sepharose and dextran sepharose beads, respectively. 2 µg of purified MBP-PTPN22 was incubated with glutathione sepharose beads bound GST or GST-mSIN. The GST-pulldowns were resolved on SDS-PAGE and analysed by immunoblotting with anti-MBP antibody to check for the interaction. Recombinant protein expression was shown by Coomassie staining. ( E ) Lentiviral transduction with control shRNA or PTPN22 shRNA was performed in HCT116 cells. Knockdown of PTPN22 was verified by examining the expression levels of PTPN22 and GAPDH mRNAs by reverse transcription polymerase chain reaction (RT-PCR). ( F ) Control or PTPN22 depleted HCT116 cells were co-transfected with SFB-mSIN and Myc-RICTOR. At 48 h post-transfection, cells were lysed in 0.3% CHAPS buffer and lysates were pulldown using S-protein agarose beads. The interactions were detected by immunoblotting with anti-Myc antibody. ( G ) Immunoprecipitation (IP) with control IgG or anti-RICTOR antibody was performed with extracts derived from either control sgRNA or PTPN22 knockout HCT116 cells. Endogenous association of RICTOR with mSIN were analysed by immunoblotting with anti-mSIN antibodies. Due to low expression level of PTPN22 in HCT116 cells, endogenous PTPN22 in input sample was shown by immunoprecipitating PTPN22 from cell extracts using its antibody. ( H , I ) Bacterially purified GST-mSIN immobilized on glutathione sepharose beads were incubated with the purified SFB-RICTOR, either in the presence of recombinant PTPN22 or equal volume of corresponding buffer. The interaction of mSIN-RICTOR was assessed by immunoblotting with anti-Flag antibody. GST-protein was used as a negative control ( H ). Individual data points for relative RICTOR bound to mSIN were plotted as graph from three independent experiments ( I ). ( J ) Jurkat cells treated either with phorbol 12-myristate 13-acetate (PMA, 1 µM) or Dimethylsulphoxide (DMSO) were lysed in 0.3% CHAPS buffer and immunoprecipitates from control IgG or anti-RICTOR antibody were analysed for the presence of PTPN22, and mTOR components by immunoblotting with their respective antibodies. ( K ) Immunoblot (IB) analysis of whole cell lysates derived from Jurkat cells treated with DMSO, PMA (1 µM), PTPN22-IN-1 (1.4 µM) or PMA treatment followed by PTPN22-IN-1 treatment, with indicated antibodies to determine the activation of AKT. .

Article Snippet: LentiCRISPRv2 vector encoding PTPN22 guide RNAs or a control sgRNA were co-transfected along with the psPAX2 packaging and pMD2.G envelope plasmids (Addgene, 12260 and 12259) in BOSC23 packaging cells using PEI.

Techniques: Isolation, Lysis, Immunoprecipitation, Control, Derivative Assay, Western Blot, Recombinant, Incubation, Expressing, SDS Page, Staining, Purification, Transduction, shRNA, Knockdown, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Knock-Out, Negative Control, Activation Assay