Polyclonal anti actin

Monoclonal antibodies against Snail-1, ERK1/2, phospho-ERK1/2, phospho-SMAD1-5-8, and Cav1 were from Cell Signaling Technology; monoclonal antibodies against E-cadherin, β-catenin, and γ-catenin were from BD (Becton-Dickinson Laboratories, Mountain View, CA); monoclonal antibodies against occludin was from Invitrogen (Carlsbad, CA); monoclonal antibodies against tubulin, α-SMA, α-catenin, vimentin, pan-cytokeratin, and fibronectin were from Sigma (Saint Louis, MO); monoclonal anti-N-cadherin and polyclonal anti-ZO-1 were from Zymed (Invitrogen, Carlsbad, CA); polyclonal anti-actin, -SMAD2-3, and -phospho-MEK were from Santa Cruz Biotechnology (CA); and polyclonal anti-phospho SMAD2-3 was from Biosource (Camarillo, CA). Monoclonal anti-CD54 was from Biolegend (San Diego, CA); polyclonal anti-Fsp1 was from Dako (Glostrup, Denmark); monoclonal anti-CD31 from Serotec (Oxford, UK); and polyclonal anti-Ki67 from Abcam (Cambridge, UK). Monoclonal anti-JAM-A clone BV-11 was a gift from Dr. E. Dejana (Milan Italy). Fluor 647?phalloidin and Hoechst 33342 were from Invitrogen (Carlsbad, CA). CI 1040 was from Selleck (Houston, TX).

Antibodies used in the study were from the following sources: monoclonal anti-NHE1 and monoclonal anti-α₁ subunit of the Na/K-ATPase (Millipore, Billerica, MA), polyclonal anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA). Polyclonal rabbit anti-NaPi-IIa, anti-NaPi-IIb, anti-NaPi-IIc, anti-Pit-1 and anti-Pit-2 were characterized and validated previously [16 (link)–18 (link)]. Immunoblotting was performed as described [36 (link)].

The following primary antibodies were used: Polyclonal rabbit anti-NaPi-IIa 1:2,000, obtained from Prof. Biber J (University of Zurich)^{58 (link)}. Polyclonal anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, 1:5000). FGFR1 mAb 9740: Cell Signaling (1:1000). Klotho MAb KM2076: Vendor Transgenics (1:2000). Secondary antibodies used were peroxidase-conjugated (Sigma-Aldrich, St. Louis, MO, USA; 1:20000).

The culture medium for the cells has been previously described [11 (link)]. Polyclonal anti-claudin-5 and anti-occludin antibodies were purchased from Zymed (San Francisco, CA, U.S.A). Polyclonal anti-actin, anti-beta tubulin, anti-p65 subunit of NFκB antibodies, monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) and anti-melanoma cell-adhesion molecule (MelCAM) antibodies were obtained from Santa Cruz (Santa Cruz, CA, U.S.A). Polyclonal anti-vascular cell adhesion molecule-1 (VCAM-1) antibodies were purchased from R&D systems (Minneapolis, MN, U.S.A). Fingolimod and Fingolimod-phosphate were provided by Mitsubishi Tanabe Pharma (Osaka, Japan).

Total protein extraction was carried out by homogenizing 10⁶ cells in 200 µL lysis buffer (10 mmoL/L Tris (pH 7.5), 5 mmoL/L MgCl₂, 10 mmoL/L NaCl, 0.5% NP-40, protease and phosphatase inhibitors, Sigma); 20 μg of total proteins were resolved by SDS-PAGE according to standard methods. Proteins were detected with anti-HIF-1α (1/500e; BD Biosciences, Allschwill, Switzerland), anti-HIF-2α (1/500; Abcam), polyclonal anti-α/β-tubulin (1/2000; Cell Signaling, Leiden, The Netherlands), polyclonal anti-actin (1/20000; Santa Cruz Biotech, Heidelberg, Germany), anti-S6 ribosomal protein (1/1000; Cell Signaling) and anti-phospho-S6 ribosomal protein (1/1000; Cell Signaling, Leiden, The Netherlands).

Immunoblotting was performed essentially as described [70 (link)]. Cell lysates were separated under reducing conditions on 15% SDS-polyacrylamide gels and electroblotted to a polyvinylidene difluoride membrane. After 1 h of blocking in 5% non-fat dry milk powder in TBST, membranes were incubated with primary antibodies overnight at 4°C. Polyclonal anti-actin, polyclonal anti-BCL-X_L, and monoclonal anti-NOXA antibodies were from Santa Cruz (Santa Cruz Biotechnology, Dallas, TX, USA). Polyclonal anti-BAX was purchased from Merck-Millipore (Darmstadt, Germany), monoclonal anti-BIM from Enzo Life Sciences, monoclonal anti-MCL-1 from Cell Signaling (Danvers, MA, USA), and polyclonal anti-PUMA antibody from Abcam (Cambridge, UK).