Caspase 3 19677 1 ap

KFs were treated as previously described, and total proteins were extracted using RIPA lysis buffer and quantified with a bicinchoninic acid assay kit (Sigma-Aldrich, US). Equal amounts of protein were separated by SDS-PAGE and then transferred onto PVDF membranes (Millipore, MA). After blocking with 5% nonfat milk, the membranes were incubated with the appropriate primary antibodies overnight at 4°C. On the following day, the membranes were washed three times with TBST (Solarbio, US) and then incubated with species-matched secondary antibodies. The membrane-bound proteins were detected using an enhanced chemiluminescence detection kit (Pierce, US) and quantified using ImageJ software. The primary antibodies used were as follows: (i) p-Smad2 (SAB4301424) and p-Smad3 (SAB4301416) (all from Sigma-Aldrich, US); (ii) Smad2 (12570-1-AP), Smad3 (66516-1-Ig), Col-I (14695-1-AP), Col-III (22734-1-AP), α-SMA (55135-1-AP), Bcl-2 (12789-1-AP), Bax (50599-2-Ig), caspase-3 (19677-1-AP) and β-actin (20536-1-AP) (all from Proteintech, US). β-Actin served as an internal control [20 (link)].

Primary antibodies against AEG-1/MTDH (13860-1-AP), E-cadherin (1702-1), N-cadherin (2447-1), and vimentin (2862-1) were obtained from Epitomics Inc. (Burlingame, CA, USA). Primary antibodies against poly-ADP-ribose polymerase (PARP) (BS70001), PI3K (BS3006), Bax (BS1030), Bcl-2 (BS1031), and C-myc (BS2462) were obtained from Bioworld Technology Inc. (Minneapolis, MN, USA). Primary antibodies against NF-κB-p65 (10745-1-AP), caspase-8 (13423-1-AP), caspase-9 (10380-1-AP), and caspase-3 (19677-1-AP) were obtained from Proteintech (Wuhan, China); Primary antibodies against Lamin A (sc-177452), AKT (sc-8312), p-AKT (sc-7985), ERK (sc-154), and p-ERK (sc-23759) were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., CA). The GAPDH antibody was purchased from Boster (Wuhan, China). The secondary antibodies peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (111-035-003), peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (111-035-003), Cy™ 3-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) (111-165-003), and Fluorescein (FITC)-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) (111-095-003) were obtained from Jackson Immuno Research Laboratories, Inc. (USA). Temozolomide (85622-93-1) was obtained from Meilun Biology Technology (Dalian, China).

The immunohistochemistry was performed as described by our previous study [30 (link)]. The ATP8 primary antibody (ab130441) was purchased from Abcam. The primary antibodies including Bax (50599-2-lg), CytC (10993-1-AP), Bcl2 (12789-1-AP), and caspase 3 (19677-1-AP) were provided by Proteintech. The p-PERK (bs-3330R) primary antibody was purchased from Bioss.

All primary antibodies were diluted at a ratio of 1:1000 as follows: PARP (9542S, Cell Signaling Technology), Caspase-3 (19677-1-AP, Proteintech), Cleaved Caspase-3 (9661S, Cell Signaling Technology), β-actin (60008-1-Ig, Proteintech), CDK4 (11026-1-AP, Proteintech), CDK6 (14052-1-AP, Proteintech), CyclinD1 (55506T, Cell Signaling Technology), AKT (9272s, Cell Signaling Technology), P-AKT Thr308 (9275s, Cell Signaling Technology), P-AKT Ser473 (4060S, Cell Signaling Technology). The second antibodies were diluted 1:5000 and purchased from Santa cruz (California, USA), including goat anti-Rabbit IgG (sc-2005) and rabbit anti-mouse IgG (sc-2004).

BLP (Pam3Cys-Ser-Lys4, ab142085) was purchased from Abcam (Cambridge, UK). Gram-negative S. typhimurium (CMCC50097) and Gram-positive S. aureus (ATCC6538) were obtained from the Laboratory of Pathogenic Microorganism, Southern Medical University, Guangzhou, China. Antibodies against autophagy-related proteins (Autophagy Antibody Sampler kit #4445) and chloroquine (CQ) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Bcl2 (#26593-1-AP), Bax (#50599-2-Ig), and Caspase3 (#19677-1-AP) were obtained from ProteinTech (Chicago, IL, USA). We purchased 3-methyladenine (3-MA) (#189490) from Sigma-Aldrich (St. Louis, MO, USA). The siRNA targeting Atg3, Atg7, and scrRNA were obtained from GenePharma (Shanghai, China).