Polyvinylidene fluoride membrane

Proteins were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Perkin-Elmer), and then probed with primary antibody (S4 Table) followed by the appropriate HRP-conjugated secondary antibody (1:5,000) (Agilent).

Cells were pretreated with darifenacin (0.1, 1 and 10 µM) for 60 min, followed by addition of ACh (10 µM) for 5 min. Following treatment with ACh, darifenacin or both, cells were lysed using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific) containing phosphatase and protease inhibitors. Protein concentration was quantified by a Quantipro BCA protein assay kit (Sigma-Aldrich) according to the manufacturer’s protocol. Cell extracts were separated by polyacrylamide gel electrophoresis and blotted on polyvinylidene fluoride membranes (Perkin Elmer, Boston, MA, USA). For immune detection, the following primary antibodies were used: anti-p38 (8690), anti-phospho-p38 (4511), anti-ERK1/2 (4695), anti-phospho-ERK1/2 (4370), anti-Akt (pan, 4685), anti-phospho-Akt (pan, 9018), anti-Src (2109S), anti-phospho-Src (6943S; all antibodies 1:1000, Cell Signaling, Leiden, Netherlands) and anti-β-actin (A5441, Sigma-Aldrich). Bound antibodies were visualized by luminescence imaging (Fusion FX, Vilber, Marne-la-Vallée, France) using peroxidase-conjugated secondary antibodies (A9169 and A9044, Sigma-Aldrich) and a chemiluminescent substrate system (SuperSignal West Pico PLUS, Thermo Fisher Scientific). Densitometric quantification was carried out with Image J. β-actin served as internal loading control.

As previously described, total protein was extracted from whole cells (14 (link)). Protein concentrations were measured by the Bradford method using Bio-Rad Protein Assay Reagent Concentrate (Bio-Rad, Hercules, CA, United States). Ten micrograms of protein lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were electroblotted onto polyvinylidene fluoride membranes (PerkinElmer Life Sciences, Boston, MA, United States). The details of the method used after blotting have been previously described (14 (link)). The antibodies were diluted in the appropriate buffer. Immunoblots including the loading control were visualized using a Lightning ECL Pro (PerkinElmer Life Sciences).

After the addition of 100 nM of calcitriol (Cayman Chemical, Ann Arbor, Michigan) for 48 h or transfection for 24 h (overexpression) or 72 h (RNAi), HT29 or SW480 cells were washed with PBS briefly, and the preparation of total cell lysates was as stated before [20 (link)]. Cellular proteins of the whole cell lysates were separated and resolved by SDS–PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Perkin Elmer Life Sciences, Inc., Waltham, MA). Western blotting was performed as previously described [20 (link)] with the following primary and secondary antibodies: MED28, β-catenin, and p-GSK3β (Ser9) antibodies from MilliporeSigma (Burlington, MA); fibronectin, E-cadherin, MMP2, and MMP9 antibodies from Abcam (Waltham, MA); anti-Flag-tag and anti-p21 from Proteintech Group, Inc. (Rosemont, IL); antibodies for vimentin, Snail, Slug, Twist, ZEB2, proliferating cell nuclear antigen (PCNA), α-tubulin, β-actin, vinculin, and GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-c-Myc, anticyclin D1, and goat antirabbit or antimouse-conjugated horseradish peroxidase secondary antibodies from GeneTex, Inc. (Irvine, CA).

Protein contents in whole-cell lysates were resolved using 8–12% SDS-PAGE and subsequently transferred to polyvinylidene fluoride membranes (Perkin Elmer, Santa Clara, CA, USA). Western blotting was performed [67 (link)] using antibodies obtained from the following sources: rabbit monoclonal anti-class III PI3K, anti-phospho-Aurora-A (Thr288), anti-phospho-MEK1/2 (Ser221)(166F8), anti-phospho-RB (Ser780), rabbit polyclonal anticaspase 3, anti-phospho-mTOR (Ser2448), anti-class I phospho-PI3K p85 (Tyr458)/p55 (Tyr199), mouse monoclonal anti-phospho-p44/42 MAPK (ERK 1/2) (Thr202/Tyr204), and anti-phospho-p70S6K (Thr389) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); rabbit polyclonal anti-LC3B and rabbit monoclonal anti-APG5L/Atg5 were obtained from Abcam (Cambridge Science Park, UK); mouse monoclonal anti-pan-RAS was obtained from Calbiochem (San Diego, CA, USA); and mouse monoclonal anti-β-actin was obtained from Sigma; goat antirabbit and antimouse conjugated horseradish peroxidase secondary antibodies were obtained from Millipore Corp. (Billerica, MA, USA). The whole western blots (uncropped blots) could see Figure S9.

Western blotting was performed as previously described (21 (link)). Approximately 1× 10⁶ cells were lysed in RIPA buffer (Beyotime) with phenylmethylsulfonyl fluoride (Sigma-Aldrich). The total protein concentration was determined using a BCA protein assay kit (Beyotime). Protein samples (30μg each) were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene fluoride membranes (Perkin Elmer). The membranes were blocked with 1xTris-buffered saline and Tween 20 (TBST) containing 5% blotting-grade blocker nonfat dry milk (Bio-Rad). They were incubated overnight with appropriate primary antibodies at 4°C followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:1000; Cell Signaling Technology). Subsequently, the protein bands on the blots were visualized using the Clarity Western ECL Kit (Bio-Rad) and imaged using the ChemiDoc Touch Imaging System (Bio-Rad). The primary antibodies against β-tubulin (#2146) were purchased from Cell Signaling Technology and used at 1:1000 dilution. The primary antibodies against MMP13 (ab51072, 1:1000) and aggrecan (ab3773, 1:100) were obtained from Abcam. Quantification through densitometry of protein bands was performed using the Image-Pro Plus software (Version 5.1; Media Cybernetics, Inc.). The experiment was performed triplicate.