Journal: bioRxiv

Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

doi: 10.1101/2022.01.26.477835

Figure Lengend Snippet: ( A-B ) HeLa cells expressing Flag-TRPM8 were incubated with DMSO dissolving different concentrations of PP2 (in μM, 0, 2.5, 10, 20) for 24 h before harvest, lysed and immunoprecipitated with an anti-Flag antibody. The samples were then analyzed by immunoblotting with the anti-Flag and p-Tyr antibodies to detect the level of TRPM8 phosphotyrosine. ( C-D ) Similar experiments in A and B but treatment with 10 μM PP2, 1 mM Na3VO4, or their combination. Na3VO4, sodium orthovanadate. PP2, 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl) pyrazolo[3,4-d] pyrimidine, a Src family kinases inhibitor. ( E-F ) Expression constructs for Flag-TRPM8 were co-transfected into PANC-1 cells with HA-BLK, HA-LCK, or HA-LYN, respectively. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the anti-Flag and p-Tyr antibodies to detect the level of TRPM8 phosphotyrosine. ( G-H ) Similar experiments in E and F but cells expressing with various amounts of HA-LCK. ( I-J ) Similar experiments in E and F but cells expressing with human LCK-specific siRNAs (siLCK#1 or #2) or negative scramble siRNAs. ( K-L ) HEK293T cells were co-transfected with GFP-M8C with HA-LCK or control vector, then harvested for IP with an anti-GFP antibodies, and WB assay with the anti-GFP and p-Tyr antibodies to detect the level of M8C phosphotyrosine. ( M ) Kinase assay in vitro. Purified GST alone or GST-M8C fusion proteins expressing in E.coli bacteria and HA-LCK immunoprecipitated with anti-HA antibody from HEK293T cells expressing HA-LCK constructs were mixed with or without 1 mM ATP in kinase assay buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 10 mM MnCl 2 ), and the reaction mixtures were then terminated, followed by WB assay with the indicated antibodies. ***, P < 0.001, NS, not significant. Data are presented as mean ± SEM. All studies were repeated at least three times.

Article Snippet: The compounds for 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl) pyrazolo[3,4-d] pyrimidine (PP2), sodium orthovanadate (Na3VO4), disuccinimidyl suberate (DSS), MG132, and saracatinib were obtained from Selleck.

Techniques: Expressing, Incubation, Immunoprecipitation, Western Blot, Construct, Transfection, Control, Plasmid Preparation, Kinase Assay, In Vitro, Purification, Bacteria