Mini trans blot apparatus

Skim milk samples were run on the 12% SDS-PAGE by a Electrophoresis apparatus (Bio-Rad, Hercules, CA, USA). The gels were electrotransferred onto the polyvinylidine difluoride (PVDF) using a Mini TransBlot apparatus (Bio-Rad, USA). PVDF membranes were blocked with 3% chickens serum in TBST solution (0.1 M Tris pH 7.4, 0.15 M NaCl, 0.1% Tween 20) and incubated with monoclonal anti-bovine β-casein solution at room temperature for 1 h. Subsequently, the PVDF membrane was washed and immersed in the horseradish peroxidase-coupled goat anti-mouse solution in the dark for 1 h. Finally, the membrane was visualized by diaminobenzidene solution. For ‘far-western blot’, the PVDF membrane was incubated with 10 μg/mL β-casein (C6905, Sigma Chemical Co., St. Louis, MO, USA) solution at room temperature for 1 h before the membrane incubated with monoclonal anti-bovine β-casein solution. The other steps were performed as the procedure of western blot.

Whole‐cell lysates were extracted by using radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors. Protein fractions were separated by 10% SDS‐PAGE (140 V). The resolved proteins were transferred (350 mA) to PVDF membranes (0.2 μm) using a mini‐transblot apparatus (Bio‐Rad). The membranes were blocked with 5% non‐fat milk at room temperature for 2 hours, incubated with the primary antibody at 4°C overnight and then incubated for 45 minutes with the appropriate secondary antibody. Proteins were finally detected with Luminata substrate. Quantification of the immunoblots was performed using ImageJ software. All optical density values from immunoblots were normalized to the density values acquired for GAPDH.

To confirm the expression of TCF4 and TCF4 mutants in cultured COS-7 cells, additionally to the microscopy experiments, SDS–PAGE and Western blot analysis were performed. Samples of the total protein extract were prepared by replacing the cell medium 24 h after transfection (after washing cells with PBS) with a 2x SDS gel-loading buffer^{66 (link)}, transferring extracts to Eppendorf tubes, boiling the samples for 5 min and then centrifuging the samples for 5 min (13,000 g). Proteins were separated by 10% SDS–PAGE and transferred to a Whatman PROTRAN Nitrocellulose Transfer Membrane (PROTRAN BA85, Schleicher and Schuell Pure, Sigma-Aldrich) with mini Trans-Blot apparatus (Bio-Rad). The membrane was blocked at room temperature (RT) for 1 h in 3% milk blocking buffer (milk powder; Milchpulver, blotting grade, Roth) that was prepared in PBS supplemented with 0.2% Tween 20 (Sigma). Next, the membrane was incubated overnight at 4 °C with anti-GFP polyclonal antibodies (Clontech) (diluted 1:300 with milk buffer), which cross-reacted with YFP. Secondary goat anti-rabbit antibodies coupled to horseradish peroxidase (Vector Laboratories) were added (1:10,000 with milk buffer) and incubated at RT for 2 h. Blots were developed using Pierce ECL Plus Western Blotting Substrate (Thermo Scientific) according to the manufacturer’s manual.

Endometrial and endometriotic tissues were suspended in RIPA⁺⁺⁺ buffer for Sodium Dodecil Sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Pooled samples were obtained by mixing equal protein amounts (μg of total protein) of 5 individual samples that were extracted in the same lysis buffer. The protein concentration in pooled samples was re-estimated using the appropriate quantification protocol. About 25 ng per sample were run on 12% SDS-PAGE for electrophoresis separation and then transferred to nitrocellulose membranes in a mini Trans-Blot apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were subsequently blocked with 3% w/v non-fat dry milk in 10 mMTris–HCl (pH: 7.5), 0.15 M NaCl, for 1 h at room temperature and then probed overnight at 4 °C with primary polyclonal antibody as reported in Supplementary Table S2, diluted in 1% non-fat dry milk/TTBS (TBS containing 0.2% Tween 20). After washing, membranes were incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody. Immunoreactivity was detected by using chemiluminescence reagents (HRP Immuno-Star HRP substrate kit; Bio-Rad Laboratories, Hercules, CA, USA). The chemiluminescence signals were captured using a Bio-Rad Chemi-Doc system and quantified using a PDQuest analysis software (Bio-Rad Laboratories, Hercules, CA, USA).

Cells were directly lysed and subjected to 10% SDS-PAGE. Immunoblotting was performed after transferring the proteins onto nitrocellulose membranes (Schleicher & Schuell Microscience) with a Mini Trans-Blot apparatus (Bio-Rad). After 2 h of blocking, the membranes were incubated overnight at 4°C with the following specific primary antibodies: anti-Jarid2 (Abcam), anti-p-STAT3, anti-STAT3 (Cell Signaling), and anti-β-actin Abs (Sigma-Aldrich). After the membranes were washed, subsequent incubations with the appropriate HRP-conjugated secondary antibodies were conducted for 1 h at room temperature; after extensive washing, the signals were visualized with an ECL substrate (Pierce Chemical).

Protein samples resolved on SDS/PAGE (12% gel) were transferred on to nitrocellulose membrane (0.45 µm) using Bio–Rad mini-transblot apparatus (120 V for 1 h). Following transfer, blots were incubated in blocking solution of 5% BSA for an hour and washed with TBS supplemented with 0.1% Tween 20 (TBST) four times for 15 min each. Blots were probed with primary antibodies (1:1000 dilution of anti-p-threonine (anti-p-Thr) with overnight incubation at 4°C diluted in blocking solution or for 1:3000 dilution of anti-His for 1 h at 25°C). Blots were washed four times with TBST (15 min/wash) and incubated with the horseradish peroxidase labelled secondary antibody (1:5000 dilution; anti-rabbit for anti-p-Thr and anti-mouse for anti-His antibodies). Finally, the blot(s) was developed using Luminataforte™ (Millipore) following manufacturer’s recommended protocol and signal was captured by exposing to X-ray film (Kodak, U.S.A.).