Stripping buffer

CD4⁺ naive T cells (2 × 10⁶) were lysed on ice with RIPA buffer (Thermo Scientific) supplemented with phosphatase (sodium orthovanadate) and proteinase inhibitor cocktails (Roche, 33576300) and phenylmethylsulfonyl fluoride (Santa Cruz Biotechnology). Total protein (10 µg) was separated on 4–15% gradient SDS gels and then transferred to polyvinylidene difluoride membranes. ORAI3, NUR77, and IKAROS were detected with the respective Abs. Membranes were stripped with stripping buffer (Invitrogen) and re-probed with anti-β-actin Abs. For TCR signaling experiments, CD4⁺ naive T cells or Jurkat cells stably transduced with ORAI3 shRNA, control shRNA, or ORAI3 were stimulated with AA or on immobilized 100 ng anti-CD3/CD28 Abs, lysed at indicated time points, and probed for p-CD3ζ (Y142), CD3, p-CAMKII, CAMKII, p-ERK (Thr202/Tyr204), ERK, and NUR77 by immunoblotting.

Two million CD4 T-naïve cells from from young (aged 20–35 years) or older (aged 65–80 years) healthy donors or naïve CD4 T cells transfected with siYY1, siYY1/pre-miR-181a, siYY1/siDUSP6, siYY1/siSIRT1, or control siRNA and stimulated by anti-CD3/CD28 were lysed on ice with RIPA buffer (Thermo Scientific) supplemented with the phosphatase inhibitor sodium orthovanadate and the proteinase inhibitor phenylmethylsulfonyl fluoride (Santa Cruz Biotechnology). Ten micrograms total protein was separated on 4–15% gradient sodium dodecyl sulfate gels and transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies specific for YY1, DUSP6, SIRT1, and pERK, respectively, then horse radish peroxidase-labeled secondary antibody and developed with Pierce ECL Western blotting substrate (Thermo Fisher Scientific). For loading control, the membrane was stripped with stripping buffer (Invitrogen) and re-probed with anti-β-actin antibodies. Uncropped immunoblot images are included in Supplementary Figs. 2–4.

Two million naive CD4 T cells from young or old healthy donors were treated with GSK3β inhibitors BIO (1 µM) or SB216763 (5 µM) for 48 h; in some experiments, cells were transfected with miR-181a inhibitor or miRNA inhibitor negative control before treatment. Cells were placed on ice and lysed with RIPA buffer (Thermo Scientific) in the presence of phosphatase and protease inhibitors, sodium orthovanadate, and PMSF (Santa Cruz Biotechnology). Approximately 10 µg of total protein was loaded in each well of SDS-PAGE gels and transferred to PVDF membranes. Membranes were incubated with primary antibodies specific for TCF1, DUSP6, and pERK, then HRP-labeled secondary antibody and developed with Pierce ECL Western blotting substrate (Thermo Fisher Scientific). Membranes were stripped with stripping buffer (Invitrogen) and the loading controls were re-probed with anti-β-actin antibodies.

Recombinant human proKLK7 was produced and purified from yeast cells as described earlier (Stefansson et al., 2008). The enzyme was then activated using thermolysin, and the activity was routinely tested against a fluorogenic substrate (data not shown). Quiescent cells were treated with recombinant active recombinant KLK7 at different concentrations and for various time periods as indicated in the Results section. Cells were lysed with RIPA buffer as described (Darmoul et al., 2004). Equal amounts of extracts (25 μg) were separated by SDS/PAGE and transferred onto a nitrocellulose membrane. Membranes were incubated in blocking buffer (20 mm Tris, 50 mm NaCl) containing 5% (w/v) low‐fat milk and 0.1% (v/v) Tween 20 and then probed with a monoclonal phospho‐specific antibody directed to ERK1/2 (1 : 2000) overnight at 4 °C. Membranes were stripped in stripping buffer (Invitrogen) and reprobed with a polyclonal anti‐ERK1/2 antibody (1 : 1000) that recognizes total ERK1/2 regardless of its phosphorylation state as loading controls. Proteins were revealed applying the Signal^® Chemiluminescent Substrate (Thermo Scientific) on an Image Quant imaging system.

Equal amounts of cell lysates were resolved on SDS–polyacrylamide gels and transferred to nitrocellulose membranes using a semi-dry blotter (Bio-Rad). Membranes were blocked using Tris-buffered saline with 5% nonfat milk (pH 8.0; Sigma). Blots were then probed with primary p53-DO1 antibody (Sigma) and POX antibody (ProteinTech) at a dilution of 1:5000 in blocking buffer, and subsequently by a secondary antibody conjugated to horseradish peroxidase (1:10,000). All blots were washed in Tris-buffered saline with Tween 20 (pH 8.0; Sigma) and developed using the enhanced chemiluminescence (ECL) procedure (Pierce, USA). Blots were then stripped using stripping buffer (Invitrogen) at 37 °C for 15 min and probed for GAPDH as loading control using anti-GAPDH antibody (Sigma) at a dilution of 1:5000. The blots were developed using anti-rabbit or anti-mouse secondary antibodies (BioRAD, USA) at a dilution of 1:10,000. Densitometry analyses were performed using Image studio version 5.2 software (LI-COR). Data were normalized to levels of GAPDH expression.

To evaluate protein levels, cell lysates were separated by SDS-PAGE and analyzed by Western blot as described^{10 (link)}. Briefly, cell pellets or snap-frozen tissues were homogenized in a lysis buffer containing 10 mM Tris-HCl, pH 6.8–7.5, 2 mM EDTA, 0.5% SDS, and freshly added 2-mercaptoethanol. Tissue homogenates were centrifuged at 12,000 g for 15 min at 4 °C, and the supernatants were collected. The total protein extracts were aliquoted to determine protein concentration using a protein assay dye reagent concentrate (Bio-Rad, CA, USA). About 100 μg total protein was separated by SDS-PAGE and then transferred to nitrocellulose membrane. The membranes were probed with the following antibodies against p22phox (1:500; sc-20781, Santa Cruz), GCLC (1:600; sc-28965, Santa Cruz), GR (1:500; sc-13324, Santa Cruz), and β-actin (1:10000, Sigma-Aldrich), washed with PBST and then probed with their respective secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. Autoradiography or the Odyssey Imaging System (LiCor Biosciences, Lincoln, NE) was used to visualize protein bands. Stripping buffer (Thermo, MA, USA) was used for sequential blotting and probing with other antibodies. ImageJ densitometry software was used to quantify individual bands.