Mini protean tgx stain free precast gel

Recombinant protein samples were mixed with 2× Laemmli sample buffer (Bio-Rad Laboratories Inc., Hercules, CA, USA) and separated in Mini-PROTEAN^® TGX Stain-FreeTM Precast Gels (Bio-Rad Laboratories Inc.). Samples were heated at 90 °C for 5 min and subjected to SDS–PAGE at 200 V for 30 min using PowerPac^TM HC high-current power supply (Bio-Rad Laboratories Inc.). For immunoblotting, proteins separated on the SDS–PAGE gel were transferred to Immobilon-P membranes (Millipore, Burlington, MA, USA), according to the manufacturer’s instructions. The membranes were blocked using TBS/3% bovine serum albumin (BSA), washed thrice using TBS/1% Tween 20, and incubated with mouse anti-His tag primary antibody (R&D Systems, Minneapolis, MN, USA) or hybridoma 2-8G in TBS/3% BSA for 90 min. After washing, the membranes were incubated for 30 min with anti-mouse IgG conjugated with horseradish peroxidase (HRP, Thermo Fisher Scientific), and immunoblots were developed using Amersham ECL detection reagent (GE Healthcare).

To obtain endogenous protein extracts from Jurkat cells, 2.5 × 10⁵ cells/well were seeded out in six-well plates and were transfected either with ANC or with syn-hsa-miR-34a-5p miScript miRNA Mimics (QIAGEN N.V., MIMAT0000255: 5′-UGGCAGUGUCUUAGCUGGUUGU-3′), complying with HiPerFect™ transfection reagent protocol (Qiagen). Further analysis was performed after an incubation time of 48 h. The transfected cells were lysed by 2 × lysis buffer (130 mM Tris/HCl, 6% SDS, 10% 3-Mercapto-1,2-propanediol, 10% glycerol) and sonification. Whole-cell protein extracts (15 μg) were separated by SDS-PAGE on Mini-Protean^® TGX Stain-Free^TM Precast Gels (Bio-Rad Laboratories Inc., Hercules, A, USA). Protein bands of NFATC4 and STIM1 were transfered by electroblotting to a nitrocellulose membrane (Whatman, GE Healthcare, Freiburg, Germany). For PPP3R1 analysis, protein bands were transferred to a polyvinylidene fluoride membrane. Protein bands were detected using specific monoclonal antibodies for STIM1 (anti-STIM1; Cat# 5668 S) and NFATC4 (anti-NFAT3; Cat# 2183 S) from Cell Signaling Technology, Inc. (Danvers, USA) and for PPP3R1 (Cat# MA5-23933) from Thermo Fisher Scientific (Waltham, USA). β-Actin served as loading control and was detected by monoclonal anti-beta-Actin antibody (Cat# A5441) from Sigma Aldrich (Munich, Germany). Secondary antibodies were purchased from Sigma Aldrich.

Overnight cultures were adjusted to 1 ml at an OD₆₀₀ of 1, spun down and resuspended in 100 μl 1× Laemmli-β-mercaptoethanol lysis buffer (Bio-Rad), and boiled for 5 min at 95°C. Then, 10 μl was run on Mini-PROTEAN TGX stain-free TM precast gels (Bio-Rad) in 1× TGX buffer for 40 min at 170 V. The gel was then stained using a Pro-Q Emerald 300 staining for glycoproteins kit (Invitrogen) following the procedure described by the supplier.

Cells were lysed in 100 µl of TNTE buffer supplemented with protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail (Sigma). TNTE buffer were composed of 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA and 0.5% Triton X-100. Then, 20 µg of proteins samples were denatured using NuPAGE sample reducing agent and LDS sample buffer (Invitrogen) for 5 min at 98 °C. Next, 6 µl of spectra^TM multicolor broad-range protein ladder (Invitrogen) or 20 µg of denatured protein samples were loaded on a 4–15% mini-PROTEAN^® TGX stain-free^TM precast gels (BioRad), transferred on trans-blot^® turbo^TM RTA midi nitrocellulose transfer kit membranes (BioRad). The membranes were blocked for 1 h in tris-buffered saline with Tween-20/5% milk, incubated overnight with primary antibodies and incubated 1 h with secondary antibodies. Signal was revealed with Super signal west femto maximum sensitivity substrate (Thermo scientific) for DPPA5 or clarity^TM ECL western blotting substrate (BioRad) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and imaged on a Chemidoc^TM MP system (BioRad). Primary and secondary antibodies are listed in Supplementary Table 2. The stain-free blot image and the uncropped blot images can be found in Supplementary Fig. 6.