Mouse anti p p65

Snap frozen tissues from Ercc1^−/Δ and WT mice were lysed in RIPA lysis and extraction buffer (Thermo Fisher). Protein concentration was determined using BCA protein assay kit (Thermo Fisher). Equal amounts of protein were loaded onto SDS‐PAGE polyacrylamide gels then transferred to 0.2 µm pore size nitrocellulose membranes (Bio‐Rad). Membranes were blocked with 5% BSA in PBS‐Tween for 1 h at room temperature then incubated with primary antibodies overnight at 4℃. Membranes were then probed with secondary antibodies at room temperature. Protein expression was measured by fluorescence using iBright™ FL1000 Imaging System. The density of each blot was quantified by using ImageJ (NIH) normalized to GAPDH. The following primary antibodies were used in this study: rabbit anti‐GAPDH (Cell Signaling Technology, 2118), rabbit anti‐p16 (Santa Cruz, sc‐1207), rabbit anti‐p21 (Abcam, ab7960), rabbit anti‐p‐p65 (Cell Signaling Technology, 3033), mouse anti‐p‐p65 (Cell Signaling Technology, 6956), rabbit anti‐Pai‐1 (Santa Cruz, sc‐8979), rabbit anti‐COX2 (Cell Signaling Technology, 12282). The following secondary antibodies were used in this study: goat anti‐rabbit IgG (H+L) Alexa Fluor Plus 488 (Thermo Fisher, A32731), goat anti‐mouse IgG (H+L) Alexa Fluor 633 (Thermo Fisher, A21052).

The antibodies used were as follows: rat anti-Ki67 (#14-5698-82, Thermo Fisher Scientific, 1:500), rabbit anti-Olig2 (#AB9610, Millipore, 1:400), rat anti-MBP (#MCA409S, Bio-Rad Laboratories, 1:500), mouse anti-CC1 (#ab16794, Abcam, 1:200), goat anti-PDGFRα (#AF1062, R&D Systems, 1:100), rabbit anti-PDGFRα (#3164 S, Cell Signaling Technology, 1:1000), mouse anti-P65 (#6956 S, Cell Signaling Technology, 1:1000), mouse anti-p-P65 (#3036 S, Cell Signaling Technology, 1:1000), mouse anti-Actin (#M1210-2, HUABIO, 1:5000), mouse anti-Tubulin (#M1305-2, HUABIO, 1:5000), mouse anti-RIPK1 (#610458, BD, 1:1000), rabbit anti-p-RIPK1 (#65746, Cell Signaling Technology, 1:1000). Secondary antibodies: Cy^TM3 affinipure donkey anti-rat IgG (H + L) (#712-165-153, 1:400), Cy^TM3 affinipure donkey anti-mouse IgG (H + L) (#715-165-151, 1:400), Alexa Fluor 488 affinipure donkey anti-rabbit IgG (H + L) (#711-545-152, 1:400) and Alexa Fluor 488 affinipure donkey anti-goat IgG (H + L) (#705-545-003, 1:400) were purchased from Jackson ImmunoResearch; Goat anti-mouse IgG (H + L) conjugated with HRP (#31430, 1:20000), goat anti-rabbit IgG (H + L) conjugated with HRP (#31,460, 1:20000) were purchased from Thermo Fisher Scientific.