Anti gapdh sc 293335

EtNBSe was synthesized by the Department of Chemistry of Central South University (Changsha, People’s Republic of China) using the synthetic method reported in our previous study.14 (link) Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The anti-GRP78 (sc-13968), anti-GADD153 (sc-7351), anti-ATF4 (sc-22800), anti-Beclin-1 (sc-48341), anti-LC3-I (sc-134226), anti-LC3-II (sc-28266), anti-Lamp (sc-5570), anti-PERK (sc-13073), anti-p-PERK (sc-32577), anti-eIF2α (sc-11386), anti-p-eIF2α (sc-12412), and anti-GAPDH (sc-293335) antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The anti-IRE1 (ab-37037) and anti-p-IRE1 (ab-124945) antibodies were purchased from Abcam (Cambridge, MA, USA). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT; M2128) was purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Detailed information about the antibodies is listed and characterized in Table 1. Salubrinal (sc-3506) and 4-phenylbutyrate (4-PBA; sc-200652) were purchased from Santa Cruz Biotechnology Inc. The autophagy activator rapamycin (#9004) was purchased from Cell Signaling Technology (Shanghai, People’s Republic of China).

A total of 2×10⁴ cells were incubated into 90 mm culture dishes. After 5 days, cells on coverslips were harvested and fixed with PBS containing 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with blocking buffer (Abcam) for 30 min at room temperature. The cells were then incubated at 4°C overnight with the following primary antibodies: Anti-RhoA (sc-418; 1:500; Santa Cruz Biotechnology, Inc.), anti-ROCK1 (sc-17794; 1:500; Santa Cruz Biotechnology, Inc.), anti-LIMK (ab119084; 1:100; Abcam), anti-MLC (ab137063; 1:500; Abcam), anti-p-MLC (ab2480; 1:500; Abcam) and anti-GAPDH (sc-293335; 1:500; Santa Cruz Biotechnology, Inc.). Subsequently, the cells were incubated with streptavidin antibodies (cat. no. 21851; 1:1,000; Thermo Fisher Scientific, Inc.) at room temperature for 30 min, followed by staining of the nuclei with DAPI (Thermo Fisher Scientific, Inc.). Images were acquired using a Nikon Eclipse E600 (Nikon Corporation, Tokyo, Japan).