Rabbit anti human gapdh

Cell debris and lysates were centrifuged at 12000 r/min for 15 min. After collecting the supernatant, the protein concentration was determined according to the instructions of a bicinchoninic acid assay kit. A 12% gel was prepared and 30 μg of protein was loaded into each lane. Proteins were subsequently transferred to polyvinylidene fluoride membranes, which were blocked in 5% skimmed milk powder in phosphate-buffered saline containing Tween 20 (PBS-T), placed on a horizontal shaker, and sealed for 1 h. Next, membranes were incubated with mouse anti-human E-cadherin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-human α-SMA (1:500, Santa Cruz Biotechnology), rabbit anti-human Snail (1:1000, Santa Cruz Biotechnology), and/or rabbit anti-human GAPDH (1:1000, Santa Cruz Biotechnology) antibodies at room temperature for 2 h, followed by 4° C for 12 h. Subsequently, membranes were washed three times (10 min each) with PBS-T on a decolorizing shaking bed, followed by incubation with appropriate secondary antibodies in a horizontal shaking bed at room temperature for 1 h. Membranes were analyzed according to instructions of an enhanced chemiluminescence kit (Bio-Rad, Hercules, CA, USA) [32 (link)].

Rabbit anti-human ADAM17 (C-terminus), and mouse anti-human CD9 were obtained from Invitrogen (Frankfurt, Germany). Rabbit anti-human GAPDH was purchased from Santa Cruz Biotech (Dallas, TX, USA), mouse anti-human JAM-A and mouse anti-human Flotilin-1 from BD Biosciences (Heidelberg, Germany), and peroxidase-conjugated anti-mouse and anti-rabbit IgG secondary antibodies from GE Healthcare (Chicago, IL, USA). TAPI-1, active-site inhibitor of ADAM17, was from Merck Millipore (Darmstadt, Germany) and human CCL2, a monocyte chemoattractant, was from Peprotech (Rocky Hill, NJ, USA). For more details see Table 1.

After treatment, sponges containing cells were rinsed once with ice-cold PBS, crushed and total proteins were extracted from cells using the RIPA-lysis buffer with a protease inhibitor cocktail. Protein concentration was assessed according to the Bradford colorimetric procedure (Bio-Rad SA). Then, 20 μg of total proteins were separated in 10% polyacrylamide gels containing 0.1% SDS and transferred to a polyvinylidene difluoride membrane (PVDF, Millipore). Unspecific binding sites of the membrane were blocked with 10% non-fat milk powder in Tris-buffered saline with 0.1% Tween (TBST) for 2 h. Then, membranes were incubated overnight at 4 °C with rabbit anti-human type I collagen (Novotec), rabbit anti-human type II collagen (Novotec) or rabbit anti-human GAPDH (Santa Cruz Biotechnology, Inc.). The following day, membranes were washed three times, followed by an incubation with HRP-conjugated goat anti-rabbit IgG antibody (Jackson Immunoresearch). Signals were visualized with the chemiluminescence method (ECL plus western blotting detection reagent+, Santa Cruz Biotechnology, Inc.) and developed on X-ray film.

Western blots were performed as described pre-viously [24 (link)]. Briefly, the total protein extract of each tissue sample or cell line was dissolved in a lysis buffer (25 mM Tris–HCl pH 7.4,1 % Triton X-100, 150 mM NaCl, 5 % EDTA, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 10 mg of aprotinin and leupeptin), separated on 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore, USA) and quantitated using a BCA protein assay kit. Equal amounts of protein (60 μg) were analyzed by immunoblotting. The antibodies used during Western blots included rabbit anti-human ERα from Proteintech, rabbit anti-human ERβ (1:500) from Proteintech, mouse anti-human MMP-1(1:400) from Santa Cruz, mouse anti-human MMP-2(1:400) from Santa Cruz, rabbit anti-human MMP-9 (1:500) from Proteintech, rabbit anti-human p38MAPK(1:500)/p-p38MAPK(1:500) and rabbit anti-human tAkt (1:500)/pAkt(1:500) from Bioworld Technology. Rabbit anti-human GAPDH (1:5000) from Santa Cruz was used as an internal control for protein loading and analysis. The protein bands were visualized using enhanced chemiluminescence (ECL) kit (Pierce, USA).