Ab60134

Minor saliva gland proteins were isolated, and western blotting was performed as follows. Proteins were extracted in the radio-immunoprecipitation (RIPA)-cocktail buffer and centrifuged. The supernatant was collected as the RIPA soluble fraction, and the pellet was washed with RIPA buffer, centrifuged and boiled with Laemmli buffer. The concentration of extracted proteins was measured using a BCA protein assay kit (Byotime, Shanghai). The following primary antibodies were used: rabbit anti-RORα (1:800, ab60134, Abcam), rabbit anti-IL-17RA (1:1000, Sangon Biotech, Shanghai), anti-RORγt (1:500, Invitrogen 14–6988-80), and mouse anti-GAPDH (1:5000, Santa Cruz Biotechnology, USA). Protein samples were separated on a 4–12% SDS-PAGE gel and electro-transferred onto a polyvinylidene fluoride (PVDF) membrane for 2 h. Membranes were blocked in blocking solution (TBST containing 5% (wt/vol) milk serum and 1% (wt/vol) BSA) for 1 h at room temperature and then incubated with primary antibodies overnight at 4 °C. Next, membranes were washed in TBST and then incubated with secondary HRP-conjugated anti-mouse (1:5000, Aspen, Wuhan) or anti-rabbit (1:5000, Aspen, Wuhan) antibodies for 1 h at room temperature. The signal was detected with enhanced chemiluminescence (ECL) Western Blotting Substrate (Thermo Scientific Pierce, P180196).

After the transfection for 48 h, total protein was extracted from cells for Western bolt assay. The primary antibodies were purchased from Abcam (RORα, ab60134; RORβ, ab228650; RORγ, ab113434). The relative expressions of RORα, RORβ, and RORγ were calculated by target protein/GAPDH using QUANTITY ONE software.

The skin tissue was cut into 0.15 cm³ pieces and placed in PBS containing 30% sucrose for dehydration. After the slicer was precooled, the tissue pieces were cut into 8 μm thick frozen sections and sealed with PBS buffer of 10% BSA (containing 0.4% Triton to increase permeability) at room temperature and dark conditions for 1 h. The slices were incubated with 0.02 mol/L primary antibody (Anti-ROR alpha, ab60134, Abcam, America) containing 0.4% Triton prepared with PBS at 4°C overnight and then incubated with 0.01 mol/L secondary antibody [Goat Anti-Rabbit IgG H&L (Alexa Fluor®) preabsorbed, ab150081, Abcam, America] containing 0.4% Triton prepared with PBS at room temperature for 1 h. Rinse with 0.01 mol/L PBS after each step. The sections were dried naturally and sealed for fluorescence microscopy to detect the location of RORα protein in skin hair follicles.

cSCCs and patient-matched normal adjacent samples were fixed in 10% neutral buffered formalin, dehydrated with gradient ethanol, hyalinized in xylene, embedded in paraffin, and made into sections. Sections (5 µm) of paraffin-embedded tissue were deparaffinized, dehydrated with gradient ethanol, and rinsed with tap water before antigen repair and serum blocking. Next, the primary anti- Rorα antibody (Abcam: ab60134, 0.5–10 μg/ml) was added for overnight reaction at 4 °C, followed by the addition of secondary antibody for 1 h of incubation. 3, 3′-Diaminobenzidine tetrahydrochloride solution was used for 15 min of coloration, followed by counterstaining with hematoxylin, dehydration with gradient ethanol, hyalinization with xylene, mounting with neutral resin, and observation under a microscope.