Anti cdkn1a p21

Equal amounts of total protein extracted from the intestinal crypt cells, were electrophoresed on 12% polyacrylamide/sodium dodecyl sulfate gel and transferred onto polyvinylidene fluoride membranes. After being blocked for 45 min, the membranes were incubated with primary antibodies for 1 h. Subsequently, membranes were incubated with horseradish peroxidase-coupled secondary antibodies for 1 h after 3 times washing with PBS. Finally, the immunoreactivity was detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA, WBKLS0500). The antibodies used were as follows: anti-HSP60 (Abcam, ab46798, 1:1000), anti-LONP1 (Cell Signal Technology, 28020, 1:1000), anti-CDKN1A/p21 (Abclonal, A2691, 1:100), anti-Phospho-eIF2α-S51 (Abclonal, AP0692, 1:1000), anti-ClpP (Abcam, ab124822, 1:1000), anti-ATF4 (Abcam, ab216839, 1:1000), anti-ATF5(Abcam, ab184923, 1:1000), anti-CHOP(Cell Signal Technology, 2895 S, 1:1000), anti-TOM20 (Proteintech, 11802-1-AP, 1:1000), anti-WNT4 (Bio-Techne, MAB4751, 1:1000), anti-SIRT7 (Abclonal, A22735, 1:1000), anti-Actin (HUABIO, ET1702-67, 1:3000), HRP Conjugated Goat anti-Mouse IgG (HUABIO, HA1006, 1:3000) and HRP Conjugated Goat anti-Rabbit IgG (HUABIO, HA1001,1:3000).

Cells were lysed with RIPA Lysis Buffer with PMSF (ST506, Beyotime Institute of Biotechnology). Proteins were quantified by BCA Protein Assay Kit (P0010, Beyotime Institute of Biotechnology). 20–80 μg of total protein was loaded onto SDS-PAGE and subsequently transferred to PVDF membranes (162-0177, Bio-Rad, Richmond, CA). After blocking with 5% nonfat milk or bovine serum albumin (BSA) in PBST, membranes were incubated with the following primary antibodies at the suggested dilutions: anti-CDKN1A/p21 (A1483, ABclonal, Cambridge, MA), anti-TP53/p53 (sc-6243, Santa Cruz Biotechnology, Santa Cruz, CA), anti-ERK1/2 (A0229, ABclonal), anti-p-ERK1/2 (AP0472, ABclonal), anti-p-Histone H2A.X (S139) (AP0099, ABclonal), anti-p-ATM (10H11.E12) (sc-47739, Santa Cruz Biotechnology), anti-p-CHK1 (Ser345) (sc-17922, Santa Cruz Biotechnology), anti-p-CHK2 (Thr68) (sc-16297-R, Santa Cruz Biotechnology), anti-PUMA (sc-374223, Santa Cruz Biotechnology), anti-TICRR (NBP2-41283, Novus Biologicals, Littleton, CO), anti-cyclin D1 (2922S, Cell Signaling Technology, Danver, MA) and anti-ACTB (AA128, Beyotime Institute of Biotechnology). Primary antibodies were detected with HRP-labeled goat anti-rabbit (A0208, Beyotime Institute of Biotechnology) or anti-mouse IgG (H+L) (A0216, Beyotime Institute of Biotechnology).