Protein Expression Analysis in Diabetic Neuropathic Pain

TLR4 and NICD1 protein levels in DRG from diabetic neuropathic pain animals and cultured DRG neurons under high glucose challenge were detected by Western blot assay. TNF-α protein levels in DRG from diabetic neuropathic pain animals were also detected by Western blot assay after Notch1 inhibitor DAPT or TLR4 inhibitor TAK treatment. The detailed Western blot assay procedures were as described in our previous study^{34 (link)}. The first antibodies used in this study were as follows: mouse anti-NICD1 monoclonal IgG (1:500, Abcam, HK), mouse anti-TLR4 monoclonal IgG (1:500, Abcam, HK), rabbit anti-TNF-α polyclonal IgG (1:1,000, Abcam, HK), and mouse anti-β-actin monoclonal IgG (1:1,000, Abcam, HK). The second antibodies were as follows: goat anti-rabbit IgG-HRP (1:3,000, Santa Cruz Biotechnology, Santa Cruz, CA) and goat anti-mouse IgG-HRP (1:3,000, Santa Cruz Biotechnology, Santa Cruz, CA). The relative levels of TLR4, NICD1, and TNF-α were normalized to control.