Cells were lysed in a buffer containing 2% SDS, 62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 5% 2-mercaptoethanol, and 0.002% bromophenol blue, and extracts were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), as previously described [42 (link)]. For Western blotting, the primary antibodies used were: anti-phospho-ERK1/2 (Thr202/Tyr204), anti-total ERK1/2, anti-PKCδ, anti-PKCα, anti-PKCε , anti-PKCη, anti-PKCζ, anti-PKCι, anti-phospho-p38, anti-total p38, anti-phospho-Akt, anti-total Akt (Cell Signaling Technology), anti-phospho-Ser299-PKCδ (Abcam), anti-actin and anti-vinculin (Bio-Rad Laboratories). As secondary antibodies we used goat anti-mouse or goat anti-rabbit antibodies conjugated to peroxidase (Bio-Rad Laboratories). Bands were visualized by enhanced chemiluminescence. Images were captured using an Odyssey Fc system (LI-COR Biosciences). Image processing and densitometry analysis were carried out using the Image Studio Lite software (LI-COR Biosciences).
Anti actin
Manufactured by Bio-Rad
Sourced in Finland
Anti-actin is a laboratory reagent used for the detection and quantification of actin, a ubiquitous cytoskeletal protein found in eukaryotic cells. It is commonly used in Western blotting, immunohistochemistry, and other analytical techniques to study the expression and localization of actin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti total akt, by Cell Signaling Technology (1 mentions)
Odyssey fc system, by LI COR (1 mentions)
Anti total erk1 2, by Cell Signaling Technology (1 mentions)
Anti total p38, by Cell Signaling Technology (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Anti pkcα, by Cell Signaling Technology (1 mentions)
Anti pkcδ, by Cell Signaling Technology (1 mentions)
Anti pkcζ, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Anti phospho p38, by Cell Signaling Technology (1 mentions)
Clarity ecl kit, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Anti axl, by Cell Signaling Technology (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by Merck Group (1 mentions)
Anti puma, by Santa Cruz Biotechnology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti adiponectin, by GeneTex (1 mentions)
Anti tubulin, by Bio-Rad (1 mentions)
5 protocols using anti actin
1
Western Blot Analysis of Signaling Proteins
2
Western Blot Analysis of Viral Proteins
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Cells were lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Deoxycholate, 1% triton X-100, 1× protease inhibitor (Roche) and boiled in a water bath for 5 min to inactivate virus. Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose. Membranes were probed with rabbit monoclonal anti-AXL (Cell Signalling), or mouse monoclonal anti-flavivirus antibodies (Millipore) and anti-ACTIN (BioRad) followed by HRP-conjugated secondary antibodies and were developed with Clarity ECL kit (Biorad) and imaged on a Fluorochem Q imager (Alpha Innotech). Images were analyzed in Adobe Photoshop and original scanned images are shown in Additional file 1 : Figure S2.
3
Protein Extraction and Western Blot
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Animals were washed off plates using M9 and snap frozen with liquid nitrogen. Pellets were sonicated in lysis buffer containing 10 mM Tris, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5 % NP-40, Halt Protease and Phosphatase Inhibitor Cocktails (Fisher Scientific, PI78440) on ice. Approximately 5 μg of total protein was loaded per well and resolved on 4–20% gradient acrylamide gels. Anti-HA antibody from rabbit (Cell Signaling Technology, 3724S) was used at 1:1,000 dilution. Anti-actin (Bio-Rad, 12004163) antibody was used at 1:4,000 dilution for loading control.
4
Western Blot Analysis of Cell Signaling
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Western blotting was performed as described [30 (link)]. Antibodies used were purchased from Bethyl (anti-p73), ProSci (anti-PUMA), Santa Cruz Biotechnology (anti-p21 (H164), anti-β-catenin (E-5), anti-Snail-1, anti-Twist), BD Transduction Laboratories (anti-E-cadherin, San Jose, CA), Sigma (anti-actin, St. Louis, MO), and BioRad (secondary antibodies against rabbit or mouse IgG conjugated with HRP, Life Science Research, Hercules, CA). Experiments were repeated at least three times.
5
Western Blot Antibody Validation Protocol
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Western Blot was performed as described previously [6 (link)]. Following antibodies were used: anti-NPR3 (EPR12716, Abcam, Berlin, Germany), anti-pAKT (Ser473; #9271, Cell Signaling, Frankfurt, Germany), anti-AKT (#9272, Cell Signaling), anti-adiponectin (#GTX112777, GeneTex, Taiwan), anti-IRAK1 (SC-5287, Santa Cruz, Heidelberg, Germany), anti-IRAK1 (#4504, Cell Signaling), anti-TRAF6 (PA29622, Invitrogen, Carlsbad, USA), anti-α-tubulin (DM1A, Merck, Darmstadt, Germany), anti-β-actin (AC-15, Sigma-Aldrich), anti-GAPDH (6C5, HyTest, Turku, Finland), hFAB rhodamine anti-GAPDH, anti-tubulin, and anti-actin (#12004168, #12004166, #12004164, BioRad, Feldkirchen, Germany).
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