Ab52628

Western blotting was performed according to our previous study [68 (link)]. Primary antibodies against MAP1LC3A (ab52628, RRID: AB_881227), CHOP (ab11419, RRID: AB_298023), XBP1s (ab220783, RRID: AB_2920809), ATF4 (ab184909, RRID: AB_2819059), Gabarap (ab109364, RRID: AB_10861928), PCNA (ab29, RRID: AB_303394), calreticulin (ab92516, RRID: AB_10562796), p-ripk3 (ab187091, RRID: AB_2619685), mlkl (ab184718, RRID: AB_2755030), p-mlkl (ab195117, RRID: AB_2768156), STAT3 (ab32500, RRID: AB_2286741) and GRP78 (ab32618, RRID: AB_732737) were purchased from Abcam; primary antibodies against ATF6 (24169-1-AP, RRID: AB_2876891), β-actin (20536-1-AP, RRID: AB_10700003), CCPG1 (13861-1-AP, RRID: AB_2074010), and STAT1 (10144-2-AP, RRID: AB_2286875) were purchased from Proteintech; primary antibodies against cleaved-caspase-3 (9661, RRID: AB_2341188), and cleaved-caspase-8 (9496, RRID: AB_561381) were purchased from Cell Signalling; and a primary antibody against LC3B (L7543, RRID: AB_796155) was purchased from Sigma (Louis, MO, USA). The secondary antibodies anti-rabbit IgG and anti-mouse IgG were purchased from Cell Signalling. β-Actin was used as a reference protein, and the protein bands were analyzed using ImageJ software.

Xenograft tumors were excised and proteins were isolated using T-Per Tissue buffer (Thermo Fisher Scientific; Waltham, MA) and homogenizer (IKA Ultra-Turrax, Fisher Scientific, Waltham, MA). Tissues were homogenized for 10 s followed by 10 min lysis, 10 min centrifugation at 13,000 rpm, and storage of supernatants at -80°C. Protein concentrations were measured using Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). For western blotting, 35 μg protein samples were separated by SDS-PAGE (12% acrylamide), transferred to PVDF membranes, and blocked with 5% milk in TBST buffer. Blots were incubated overnight with the following antibodies purchased from Abcam (Cambridge, MA) or Cell Signaling (Danvers, MA): LC3A (1:500; ab52628, Abcam), LC3B (1:500; ab168831, Abcam), mTOR (1:500; 2972, Cell Signaling). GAPDH (1:2500; ab9485, Abcam) was used as loading control and HRP-conjugated goat anti-rabbit (1:5000, ab6721, Abcam) as secondary antibody. For detection and imaging, Clarity Western ECL substrate and ChemiDoc touch imaging system (Bio-Rad, Hercules, CA) were utilized following manufacturer’s protocol.