Non fat milk

For cell lysis and total protein extraction, RIPA protein extraction buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) and protease and phosphatase inhibitor cocktails (Sigma-Aldrich) was used. Protein concentration was determined by the Bradford method (Bio-Rad Laboratories). Sixty micrograms of extracted proteins was resolved on 10% SDS-PAGE (Bio-Rad Laboratories) and transferred on PVDF membranes (Sigma-Aldrich) by semi-dry blotting (Whatman; Biometra GmbH, Göttingen, Germany). The membranes were blocked for 1 h in 5% non-fat milk (Sigma-Aldrich) and incubated at 4 °C, overnight with a primary antibody (1:200 goat polyclonal anti-WWOX, Santa Cruz Biotechnology, cat. no. sc-20529). Subsequently, the membranes were washed with Tris-buffered saline-Tween 20 (TBST) buffer and incubated with the secondary antibody (1:15,000 anti-goat IgG, Sigma-Aldrich, cat. no. A4187) conjugated with alkaline phosphatase for 1 h at room temperature, followed by washes with TBST and developed with Novex® AP Chromogenic Substrate (Life Technologies). As a reference, mouse anti-GAPDG monoclonal antibody was used (1:1000; Santa Cruz Biotechnology, cat. no. sc-59540). The relative protein amount was assessed with ImageJ (Schneider et al. 2012 (link)) based on the integrated density of the bands.

The protein concentration was quantified by BCA assay (ThermoFisher Scientific), and 20–40 µg of protein was separated in 8% PAGE gel. The proteins were transferred to a nitrocellulose membrane at 35 V for 3 h for Ca_V3.1 and Ca_V3.2 and 100 V for 1 h for pERK1/2. The protein transfer was confirmed by Ponceau staining, and the blots were washed and incubated with 5% non-fat milk (Sigma) for 1 h. The blots were incubated with primary antibodies: Ca_V3.1 (1:1000) (Alomone, Jerusalem, Israel) and Ca_V3.2 (1:500) (Alomone), pERK1/2 (1:5000) (Cell signaling, London, UK), total ERK1/2 (1:5000) (cell signaling) and beta-actin (1:5000) (Abcam, Cambridge, UK) overnight at 4 °C. The following day, the blots were washed thrice with 1 × TBS + 0.1% Tween 20 (TBS-T) for 10 min and then incubated with anti-rabbit secondary antibody conjugated with horse radish peroxidase for 1 h at room temperature. The blots were washed thrice with 1 × TBS-T and incubated with ECL substrate (Fisher scientific) for 5 min and exposed to X-ray film. In order to probe for total ERK1/2 and beta-actin, the blots were stripped with stripping buffer (Sigma) for 30 min at RT under dark. Protein band density was quantified using Image Studio Lite software.

Immulon 4 HBX 96-well microtiter plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 250 ng/well of goat anti-human IgA (Bethyl Laboratories, #A80-102A) in PBS (pH 7.4). Well contents were discarded and blocked with 200 µL of 5% non-fat milk (AmericanBio) in PBST for one hour at RT. After blocking, 50 µL of serum samples diluted (starting at 1:256 and serially diluted three-fold) with 2.5% non-fat milk in PBST was added to each well and incubated at RT for 2 h. After washing with PBST three times, 50 μL of HRP-labeled goat anti-human IgA antibody (Bethyl Laboratories, #A80-102P) diluted to 1:10,000 with 2.5% non-fat milk in PBST was added to each well, and incubated at RT for 1 h. After washing with PBST three times, 100 μL of SIGMAFAST o-phenylenediamine dihydrochloride substrate solution (Sigma-Aldrich) was added to each well for reaction at RT for 10 min. The reaction was stopped by the addition of 50 μL of 3 M hydrochloric acid (HCl). Optical density at 490 nm was measured using a Synergy 4 (BioTek) plate reader. Purified human plasma IgA (EMD Millipore, #401098) was diluted (starting at 500 ng/mL and serially diluted two-fold) and was used as standard. A five-parameter logistic fit was conducted on the standard curve and IgA concentrations within saliva samples were calculated. The assay was done once per sample due to the limited sample amount.

Total protein was extracted in RIPA lysis buffer. Proteins extracted from cells were resolved using 10% sodium dodecyl sulfate–polyacrylamide (SDS–PAGE) gel electrophoresis, then transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA), blocked in 5% non-fat milk (Sigma–Aldrich, St Louis, MI, USA) for 2 h, and blotted with primary antibody (anti-NEDD4, Cell Signaling Technology, Danvers, MA, USA, 1:1000; anti-IGF-1R, Abcam, 1:1000; anti-PTEN, Cell Signaling Technology, 1:500; anti-p-Akt^ser473, Cell Signaling Technology, 1:2000; anti-GAPDH, Cell Signaling Technology, 1:2000; anti-β-actin, Sigma–Aldrich, 1:5000) overnight at 4 °C. The next day, membranes were incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. Blots were visualized with an ECL detection kit (Millipore) and analyzed using Image J software.

Cells were washed twice with cold phosphate-buffered saline, then lysed with RIPA lysis buffer containing protease inhibitor and shaken for 15 minutes on ice. Protein samples were subjected to centrifugation at 12,000 ×g for 15 minutes, and the supernatants were collected. Protein concentration was determined using bicinchoninic acid protein assay kit (Beyotime). Samples containing equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a 0.22-μm polyvinylidene difluoride membrane. After blocking with 5% non-fat milk (Sigma, St. Louis, USA), the membranes were incubated with a primary antibody (PPARγ: ab191407, Abcam, Cambridge, UK; LPL: ab172953, Abcam; α-tubulin: T9026, Sigma) at 4°C overnight, then further incubated with the corresponding secondary antibody (170-6515 and 170-6516, Bio-Rad) according to the manufacturer’s instructions. Protein bands were quantified using the Amersham Imager system.