Total proteins were extracted according to a previously described protocol [62 (link)]. Equivalent amounts of protein were separated by 10% SDS–PAGE (20 μg per lane) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% skim milk (dissolved in TBST buffer), the membranes were incubated with primary antibody overnight at 4 °C, washed three times with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for one hour at room temperature. The primary antibodies were listed as follows: NFATc1 (1:1000; Santa Cruz, #sc-7294), CTSK (1:1000; Santa Cruz, #sc-48353), p-P65 (1:1000; CST, #3033), P65 (1:1000; CST, #8242), IkBα (1:1000; CST, #4814), c-Fos (1:1000; CST, #5348), GAPDH (1:3000; Abway, #AB0036), NRP1 (1:1000; Abcam, #81321), β-actin (1:3000; Abway, #AB0011). Finally, antibody activity was detected using ECL hypersensitive chemical luminescence reagents (Yeasen, Shanghai, China) and imaged by an Invitrogen iBright 1,500 system (Thermo Fisher Scientific Scientific). The band density was quantified using ImageJ software.
Free full text: Click here
