Immunoblotting was performed as previously described.19 (link),34 (link) The β-actin antibody was used to normalize protein expression. For CHX chase assays, cells were treated with 10 μmol CHX for 24 hours after transfection and collected at the indicated time points, and cell lysates were subjected to immunoblotting. Anti-HDLBP (Proteintech, 15406-1-AP, Wuhan, China, 1:800), Anti-RAF1 (Cell Signaling Technology, #53745, Danvers, MA, 1:800), Anti-β-actin (Proteintech, 66009-1-Ig, 1:2500), Anti-Lamin B1 (Proteintech, 12987-1-AP, 1:2500), Anti-Flag (Proteintech, 20543-1-AP, 1:2000), Anti-HA (Proteintech, 51064-2-AP, 1:3000), Anti-His (Proteintech, 66005-1-Ig, 1:2500), Anti-Myc (Proteintech, 16286-1-AP, 1:800), Anti-TRIM71 (Proteintech, 55003-1-AP, 1:800), Anti-NEDD4L (Proteintech, 13690-1-AP, 1:800), Anti-p-MEK (Cell Signaling Technology, #8727, 1:500), Anti-MEK (Cell Signaling Technology, #9154, 1:500), Anti-p-ERK (Cell Signaling Technology, #4695, 1:500), Anti-ERK (Cell Signaling Technology, #4377, 1:500), Anti-MEKK1 (Proteintech, 19970-1-AP, 1:500), Anti-RAF1S338 (Cell Signaling Technology, #9427, 1:500), Anti-RAF1S259 (Cell Signaling Technology, #9421, 1:500), Anti-RAF1S621 (Abcam, ab157201, Cambridge, UK, 1:500), Anti-RAF1Y341 (Abcam, ab59223, 1:500), and Anti-RAF1S289/296 (Cell Signaling Technology, #9431, 1:500) were used.
Anti nedd4l
Manufactured by Proteintech
Sourced in United States
Anti-NEDD4L is a primary antibody that specifically recognizes the NEDD4L protein. NEDD4L is an E3 ubiquitin-protein ligase that plays a role in the regulation of various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by Proteintech (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti lamin b1, by Proteintech (1 mentions)
Anti myc, by Proteintech (1 mentions)
Anti raf1, by Cell Signaling Technology (1 mentions)
Anti hdlbp, by Proteintech (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti mek, by Cell Signaling Technology (1 mentions)
Anti p mek, by Cell Signaling Technology (1 mentions)
Anti ha, by Proteintech (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti β actin, by Thermo Fisher Scientific (1 mentions)
Anti 53bp1, by Novus Biologicals (1 mentions)
Anti rhob, by Santa Cruz Biotechnology (1 mentions)
Anti rpa32, by Fortis Life Sciences (1 mentions)
Np40 cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti flag hrp, by Merck Group (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Anti sp1, by Proteintech (1 mentions)
3 protocols using anti nedd4l
1
Immunoblotting and CHX Chase Assays
2
Protein Extraction and Western Blotting
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Cells were lysed using NP40 cell lysis buffer (Invitrogen, Burlington, Canada FNN0021), supplemented with Halt protease inhibitor cocktail (Thermo-Scientific PI87786). The cell lysates were diluted with PBS to 25 mg/mL, and concentration was measured using DeNovix spectrophotometer. In general, 50–100 μg of protein samples was loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 4–15% gradient gel (Bio-Rad 4561086) and transferred onto 0.2 μm PVDF membranes (Cytiva, Toronto, ON, Canada 10600021). The membranes were blotted by the indicated antibodies: anti-FLAG-HRP (Millipore Sigma, Oakville, ON, Canada A8591), anti-53BP1 (Novus Biologicals, Toronto, ON, Canada NB100-304), anti-RFWD3 (Abcam ab138030), anti-NEDD4L (Proteintech, Rosemont, USA 10091-010), anti-RhoB (Santa Cruz, Dallas, TX, USA sc-108), anti-RPA32 (Bethyl Laboratories, Montgomery, USA A300-244A), anti-Ub (clone FK2, Millipore 04-263), and anti-β-actin (Thermo Scientific, Mississauga, ON, Canada MA515739). SuperSignal West ECL (Thermo Scientific, Mississauga, ON, Canada PI34579) was used to image the bands with Bio-Rad ChemiDoc XRS+ Imaging system.
3
Integrin Signaling Pathway Analysis
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Immunoblotting was conducted as reported previously. Briefly, cell samples were lysed in lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; and the protease inhibitor, 1 mM PMSF), and tissue samples were prepared in tissue protein extraction reagent (Thermo Fisher Scientific). Protein (40 μg) was processed for the analysis. The antibodies used for the analysis were as follows: anti- Integrin α5 (1:200, Santa Cruz Biotechnology), anti-Integrin β3 (1:1000, Cell Signaling Technology), anti-SP1 (1:1000, Proteintech), anti-p-MEK1/2 (Ser218/222, 1:1000, Cell Signaling Technology), anti-MEK1/2 (1:1000, Cell Signaling Technology), anti-p-NEDD4L (Ser448, 1:1000, Abcam), anti-NEDD4L (1:1000, Proteintech), anti-ubiquitin (1:500, Santa Cruz Biotechnology), anti-GAPDH (1:1000, Beyotime).
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