Mouse anti gapdh

Western blotting was performed as previously described [54 (link)]. The samples were lysed in lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 100 mM NaF, 1 mM vanadate, and protease inhibitors) to lyse cells and fresh brain tissue. The primary antibodies used were: mouse anti-DCC (1:500, #554223, BD, San Jose, CA, USA ), mouse anti-His (1:1000, TA-02, ZSGB-BIO, Beijing, China), mouse anti-myc (1:1000, 22E8, Sungene Biotech, Tianjin, China), mouse anti-Netrin-1 (1:1000, ALX804838, Enzo Life Sciences, Farmingdale, New York, USA ), mouse anti-flag (1:1000, F3165, Sigma Aldrich, St Louis, MO, USA), rabbit anti-phospho-FAK(pTyr861) (1:1000, F9176, Sigma Aldrich, St Louis, MO, USA), rabbit anti-FAK (1:200, sc-557, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-phospho-SRC (Tyr418)(1:1000, Invitrogen, Carlsbad, CA, USA), rabbit anti-SRC Family (1:1000, #2123, CST, Danvers, MA, USA), mouse anti-GAPDH (1:5000, TransGene Biotech, Beijing, China), mouse anti-beta actin (1:3000, A5441, Sigma Aldrich, St Louis, MO, USA), rabbit anti-phospho-ERK(Thr202/Tyr204) (1:1000, #9101, CST, Danvers, MA, USA), rabbit anti-ERK (1:1000, #4695, CST, Danvers, MA, USA), HRP-conjugated secondary antibody against mouse, goat or rabbit were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

Cell lysates were separated on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) and incubated with indicated primary antibodies at 4 °C for 12 h. After washing three times with TBS-T, membranes were incubated with secondary antibodies at RT for 1 h. ECL western blot substrate was used to visualize protein bands. Primary and secondary antibodies: rabbit anti-MLKL (Proteintech, 21066-1-AP), rabbit anti-p-MLKL (T357/S358) (Abcam, ab187091), rabbit anti-RIPK1 (CST, 8737), rabbit anti-RIPK3 (Abcam, ab72106), rabbit anti-ICAM1 (CST, 4915S), rabbit anti-VCAM1 (CST, 13662S), mouse anti-GAPDH (TransGen, HC301-02), mouse anti-Tubulin (TransGen, HC101-02), horseradish peroxidase (HRP) conjugated donkey anti-rabbit and HRP conjugated donkey anti-mouse were obtained from Jackson lab.

Mice were deeply anesthetized with isoflurane and L4–L5 DRGs were dissected and placed in RIPA assay lysis buffer (C50008, Sangon Biotech). Protein concentration was measured using a bicinchoninic acid (BCA) assay (P0010, Beyotime Biotechnology, China). Proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes. PVDF membranes were blocked for 90 min at room temperature using 5% BSA in TBS-T and subsequently probed with the following primary antibodies at 4°C overnight: rabbit anti-PTPRD (1:1000, Novus, NBP2-94767), rabbit anti-STING (1:1000, CST, D2P2F), or mouse anti-GAPDH (1:1000, TransGen Biotech). The membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1000, Beyotime) for 120 min at room temperature. Finally, images were acquired using a GS800 Densitometer Scanner. The optical density of protein bands was quantified using ImageJ, with GAPDH used as the loading control to normalize protein expression levels. Each experiment was repeated three times.