Anti hsc70 10654 1 ap

Cells were collected and lysed using RIPA buffer (150 mM NaCl, 1% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris-HCl pH 8) supplemented with protease and phosphatase inhibitors (Roche, Switzerland) and total protein concentration were measured by BCA assay. Whole-cell lysates (~20–30 μg of proteins) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblotting was performed using the following primary antibodies: anti-p38 (#8690), anti-phospho p38 (T180/Y182 - #4511), anti-p53 (#2524), anti-phospho pS6 (S235/236 - #4058), anti-Akt (#9272), anti-phospho-Akt (S473, #4060), anti-caspase-3 (clone 8G10; #9665), anti-HK2 (#2867), anti-HER2 (#2165), anti-mTOR (#2983), anti-phospho-mTOR (S2448—#5536), anti-PARP (#9542) from Cell Signaling Technology (Massachusetts, USA); anti-LC3 (L7543) from Sigma-Aldrich; anti-Actin (sc-8432), anti-GAPDH (sc-166545), anti-IκB-α (sc-371), anti-Lamp2 (sc-18822) and anti-tubulin (sc-73242) from Santa Cruz Biotechnology (Texas, USA); and anti-Hsc70 (10654–1-AP) from Proteintech (Illinois, USA).

Cell lysates were prepared using ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 1 mM C₃H₇Na₃O₆P, 1 mM NaF, 1 mM Na₄O₇P₂, 1 mM Na₃VO₄, and 5 mM PMSF) supplemented with protease/phosphatase inhibitors (Roche, Basel, Switzerland). Western blot experiments were conducted as described previously [60 (link)]. The blots were probed with the following primary antibodies: Anti-HER2 (#2165), anti-HSP90 (#4877), anti-phospho-HER2 (Tyr1221/1222, #2243), anti-phospho-AKT (S473, #4060), anti-phospho-ERK1/2 (Thr202/Tyr204, #4370), anti-phospho-NF-κB (Ser536, #3033), and anti-phospho-SRC family (Tyr416, #2101) were obtained from Cell Signaling Technology (CST, MA, USA). Anti-HER2(sc-08) and anti-N-cadherin (sc-271386) were obtained from Santa Cruz Biotechnology (TX, USA). Anti-ubiquitin (10201-2-AP), anti-HSP70 (10995-1-AP), and anti-HSC70 (10654-1-AP) were obtained from Proteintech (Wuhan, China). Anti-E-Cadherin (610181) was obtained from Becton, Dickinson and Company (BD Biosciences, NJ, USA). Anti-Vinculin (V9131) was obtained from Sigma Aldrich (MO, USA). Fluorescent-labeled secondary antibodies against mouse IgG and rabbit IgG were used (Li-COR, Nebraska, USA). Western blots were imaged and quantified by Odyssey Infrared Imaging System (Li-COR, Nebraska, USA).