Anti hspb1 d6w5v rabbit mab

Four pairs of cancerous tissues and matched noncancerous normal liver tissues were collected from HB patients who underwent surgery at the Department of Hepatobiliary Surgery of the Shanghai Children’s Medical Center. Patients did not receive chemotherapy or radiotherapy before admission. This study was approved by the Ethics Committee of the Shanghai Children’s Medical Center, and written informed consent was obtained from each patient. The detailed information of these patients is listed in Supplementary Table S2.
Immunohistochemical staining was performed as previously described.^{47 (link)} 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), and anti-HSPB1 (D6W5V) rabbit mAb (#95357, Cell Signaling Technology, MA, USA).

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).

Tissues and cells were lysed in IP lysis buffer (Beyotime, Jiangsu, China), and 500 μg samples of total protein were incubated with specific antibodies and protein A/G PLUS agarose beads (Santa Cruz, CA, USA)) overnight at 4 °C. The beads were washed with IP lysis buffer, and western blotting was then performed. The antibodies used were anti-Myc (9B11) mouse mAb (#2276S, Cell Signaling Technology, MA, USA), anti-HSPB1 (D6W5V) rabbit mAb (#95357, Cell Signaling Technology, MA, USA), anti-NPM1 rabbit pAb (10306-1-AP, Proteintech, IL, USA) and anti-HSPE1 rabbit mAb (ab108600, Abcam, MA, USA).