Ab3350

P1 pups were intraperitoneally injected with BrdU (100 mg/kg; Sigma-Aldrich). Their pancreata were fixed in 4% PFA in PBS for 12 h at 4 °C followed by dehydration, paraffin embedding, and histologically analyzed as described previously (Lee et al, 2007 (link); Wei et al, 2014 (link)). For β-cell proliferation analysis in P1 pups every other section was labeled with guinea pig anti-insulin (1:200; DAKO A0564) and mouse anti-BrdU (1:500 Sigma B2531) antibodies, followed by fluorochrome-coupled secondary antibodies (Millipore) and DAPI counterstaining (Abcam). β-cell proliferation was quantified by counting the number of BrdU/insulin-positive cells over the total number of insulin-positive cells using ImageJ software. An average of 2000 insulin-positive cells per specimen was counted. Bmal1 and Clock levels were assessed on pancreas sections of P1 pups following co-labeling with rabbit anti-Bmal1 (1:500 Abcam ab3350) or rabbit anti-Clock (1:1000 Abcam ab3517) antibodies and a guinea Pig anti-insulin (1:800 DAKO A0564) antibody while Pck1 levels were assessed using a rabbit anti-PCK1 (1:400 Abcam ab70358) antibody, followed by fluorochrome-coupled secondary antibodies (Invitrogen A21206 and A21450).

Bicinchoninic acid assay (BCA assay) relative protein quantification was used for normalization of sample loading. Proteins with 6 μg (EGFR, CD81, FGF-2, Flottin-1, Grp94, HSA), 8 μg (securin) or 10 μg (NLGN3) were loaded on per channel. Samples were separated using the assay of vertical slab polyacrylamide gel electrophoresis. After that, proteins were transferred onto polyvinylidene fluoride membranes (PVDF Invitrogen) using wet electrophoretic transfer. The membranes were blocked with 5% (w/v) skim milk and then incubated with primary antibody in 3 mL primary antibody dilution buffer. Next, the membranes were washed three times for 5 min each with 5 mL TBST followed by incubation with secondary IgG and HRP-linked antibody.
Western blot antibodies are listed below: Anti-EGFR, Abcam, ab52894, 1/5000. Anti-CD81, CST, 10037S, 1/1000. Anti-FGF2, Abcam, ab16828, 2 μg/ mL. Anti-Securin, Abcam, ab3350, 1/1000. Anti-HSA, Biorbyt, orb24991, 1/1000. Anti-Grp94, Sino Biological, 106461, 1/1000. Anti-Flotillin 1, Abcam, ab133497, 1/10000. Anti-CD81, CST, sc166029, 1/500. Anti-mouse IgG, HRP-linked Antibody, CST, #7076, 1/1000. Anti-rabbit IgG, HRP-linked Antibody, #7074, 1/1000.

Forskolin and melatonin were purchased from Sigma-Aldrich. Y-27632 and arachidonic acid (AA) were purchased from Selleck Chemicals. The following antibodies were used in this study: BMAL1 (Abcam ab3350 for immunofluorescence and Santa Cruz Biotechnology sc-365645 for western blot), aggrecan (Millipore Sigma AB1031), MMP13 (Abcam ab39012 for western blot and Proteintech 18165-1-AP for immunofluorescence), and phosphomyosin light chain 2 (Ser19; Cell Signaling Technology 3671). Horseradish peroxidase (HRP)-conjugated β-actin mouse monoclonal antibody, HRP-conjugated AffiniPure goat anti-mouse or goat anti-rabbit IgG (H + L), fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit IgG (H + L), and Cy3-conjugated AffiniPure goat anti-rabbit IgG (H + L) were purchased from Proteintech. Cell culture reagents were purchased from Gibco.