Anti myc tag 9b11 mouse mab

Tissues and cells were lysed in western lysis buffer (Beyotime, Jiangsu, China) for 30 min on ice, followed by centrifugation at 12,000×g for 15 min. Protein samples (30 μg) were separated on SDS–PAGE gels and transferred to PVDF membranes (GE Healthcare, Buckinghamshire, UK). Membranes were blocked with 5% BSA at room temperature for 1 h, followed by overnight incubation with primary antibodies at 4 °C. Membranes were washed with PBST three times, followed by incubation with the appropriate HRP-conjugated secondary antibodies (Santa Cruz, CA, USA) at room temperature for 1 h. Bands were visualized using a SuperSignal West Femto kit (Pierce, IL, USA). GAPDH was used as the loading control. The primary antibodies used were anti-OGT (D1D8Q) rabbit mAb (#24083, Cell Signaling Technology, MA, USA), anti-O-GlcNAc mouse mAb (PTM-952, PTM Bio, Zhejiang, China), anti-HSPB1 (D6W5V) rabbit mAb (#95357, Cell Signaling Technology, MA, USA), anti-Phospho-HSPB1 (Ser82) (D1H2F6) rabbit mAb (#9709, Cell Signaling Technology, MA, USA), anti-NPM1 rabbit pAb (10306-1-AP, Proteintech, IL, USA), anti-HSPE1 rabbit mAb (ab108600, Abcam, MA, USA), anti-phosphoserine/threonine/tyrosine mouse mAb (ab15556, Abcam, MA, USA), anti-Myc-Tag (9B11) mouse mAb (#2276S, Cell Signaling Technology, MA, USA) and anti-phospho-(Ser) Arg-X-Tyr/Phe-X-pSer motif rabbit Ab (#2981S, Cell Signaling Technology, MA, USA).

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 5 min and blocked with 3% bovine serum albumin (BSA) for 1 h. Cells were incubated with an anti-FLAG tag (D6W5B) rabbit monoclonal antibody (mAb) (Cell Signaling Technology, Cat No. #14793, 1:100) and anti-Myc tag (9B11) mouse mAb (Cell Signaling Technology, Cat No. #2276, 1:100) at 4 °C overnight, followed by treatment with a goat anti-rabbit IgG secondary antibody conjugated to Alex Fluor 488 (Invitrogen, Cat No. R37116, 1:200) and donkey anti-mouse IgG secondary antibody conjugated to cyanine cy5 (Jackson ImmunoResearch, Cat No. 715–175-150, 1:200).

Cells were lysed in Cell Extraction Buffer, 1 mm PMSF (P7626, Sigma-Aldrich) protease inhibitor (11836153001, Roche Applied Science), and Phosphatase Inhibitor Mixture 2/3 (Sigma-Aldrich). Two milligrams of the lysate was incubated with 2 μg of monoclonal anti-FLAG M2 antibody (F3165, Sigma-Aldrich) or anti-Myc tag (9B11) mouse mAb (2276, Cell Signaling Technology) for 2 h at 4 °C, and immunocomplexes were precipitated using protein-G and A-Sepharose beads (17-6002-35, GE Healthcare). Beads were then washed three times with extraction buffer.