Af5220

Stachydrine hydrochloride (STA, Cas: 4136-37-2) was acquired from the National Institutes for Food and Drug Control (Beijing, China). Phenylephrine (PE) was bought from Sigma-Aldrich (St. Louis, MO, USA). The remaining reagents were purchased as follows: H₂O₂ (LIRCON), NAC (N-acetyl-L-cysteine, Selleck, S1623), GSK (GSK-2795039, inhibitor of NOX2, Selleck, S1415925-18-6), LTCC (Abcam, ab253190), CaMK2 (Abcam, ab181052), P-CaMK2 (Invitrogen, PA5-37833), OX-CaMK2 (Gene Tex, GTX36254), RyR2 (Abcam, ab2868), P-RyR-2814 (Badrilla, A01031AP), P-RyR-2808 (Badrilla, 642104), NOX2 (Abcam, ab80897), p22phox (Abcam, ab75941), p67phox (Abcam, ab175293), p47phox (Affinity, AF5220), and p-p47phox (Affinity, AF3917).

Kidney tissues or cells were lysed and subsequently sonicated in PBS that contained 1% Triton X-100, 250 mmol/L phenylmethanesulfonyl fluoride, 2 mmol/L EDTA, and 5 mmol/L dithiothrietol (pH7.5). The total protein concentration was then determined by the BCA protein assay reagent kit (GBC, G3422). Thirty milligrams of protein for each sample was denatured in boiling water for 5 min then separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The blots were blocked 1 h with 5% nonfat dry milk in Tris-buffered saline, followed by incubation overnight with rabbit α-smooth muscle actin (α-SMA) antibody (Affinity, AF1032), Collagen IA (Affinity, AF7001), NLRP3 (Abcam, ab214185), Caspase-1 (Affinity, AF4005), APJ (Fitzgerald, 70R-51439), p47phox (Affinity, AF5220) or p22phox (Affinity, DF10099) at 4 °C. For β-actin, the membranes were stripped and reprobed with rabbit anti-β-actin antibody (Affinity, AF7018) or anti-GAPDH antibody (Affinity, AF7021). After being washed with Tris-buffered saline, membranes were incubated with a secondary antibody labeled with horseradish peroxidase-conjugated secondary antibody (Affinity, S0001) and visualized using enhanced chemiluminescence. The intensities of blotted bands were quantified with the software (ImageJ, free download from http://rsbweb.nih.gov/ij/).