Four days after differentiation, muscle cells cultured in 12-well plates were washed with cold PBS and lysed on ice with 200 µL of RIPA buffer with protease and phosphatase inhibitors. Protein concentrations were quantitated with the Bio-Rad protein assay, and all samples were standardized in order to load 20 µg of protein per polyacrylamide gel lane. PAGE was performed, and proteins were transferred to PVDF membranes for probing. Membranes were blocked with 5% milk powder and incubated with primary antibodies overnight at 4°C before incubation with secondary antibodies for 2 h and chemiluminescence imaging. Primary antibodies used were rabbit anti-phospho-Akt (Ser473) D9E (Cell Signaling, 4060), rabbit anti-phospho-Erk antibody (Cell Signaling, 9101), rabbit anti α-tubulin 11H10 (Cell Signaling, 2125), and mouse polyclonal anti-MLX (Abcam, ab167286; this weak-affinity antibody detects MLX upon overexpression but not its endogenous levels).
Rabbit anti phospho akt ser473 d9e
Manufactured by Cell Signaling Technology
Rabbit anti-phospho-Akt (Ser473) D9E is a primary antibody that recognizes the phosphorylated form of Akt at serine 473. It is used for the detection and quantification of activated Akt in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti pan akt 11e7, by Cell Signaling Technology (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Protein assay, by Bio-Rad (1 mentions)
Dual color protein standard, by Bio-Rad (1 mentions)
Rabbit anti p src tyr416, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho fak tyr 397, by Cell Signaling Technology (1 mentions)
Mouse anti gapdh, by Merck Group (1 mentions)
Supersignal west femto substrate kit, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti dnak 8e2 2, by Enzo Life Sciences (1 mentions)
Mouse monoclonal anti mcherry, by Takara Bio (1 mentions)
Mouse monoclonal anti ha 16b12, by Fortrea (1 mentions)
Rabbit anti rnase 7, by Merck Group (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Invitrolon pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Rabbit anti erk1 2 137f5, by Cell Signaling Technology (1 mentions)
Nupage bis tris mini gel, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
5 protocols using rabbit anti phospho akt ser473 d9e
1
Western Blot Analysis of Muscle Cells
2
Western Blot Analysis of Signaling Proteins
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Cell lysis, SDS-polyacrylamide gel electrophoresis, protein transfer and western blot were performed similarly as before31 (link). Dual Color Protein Standard (Bio-Rad, Hercules, CA) was used to determine molecular weight. Antibodies used included rabbit anti-phospho-FAK (Tyr397), (Cell Signaling Technology, Danvers, MA), mouse anti-FAK clone 4.47 (EMD Millipore, Darmstadt, Germany), rabbit anti-FoxM1 (Novus Biologicals, Littleton, CO), rabbit anti-Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology), rabbit anti-ERK total (Cell Signaling Technology), rabbit anti-phospho-AKT (Ser473) (D9E) (Cell Signaling Technology), mouse anti-GAPDH (EMD Millipore), rabbit anti-AKT total (Cell signaling Technology), rabbit anti p-Src (Tyr416) (Cell Signaling Technology) and mouse anti-total Src (Cell signaling Technology).
3
Immunoblotting with Fluorescent Antibodies
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Primary antibodies used were mouse monoclonal anti-GFP (11E5, Molecular Probes), mouse monoclonal anti-mCherry (Clontech), mouse monoclonal anti-DnaK (8E2/2, Enzo Life Sciences), mouse monoclonal anti-HA (16B12, Covance), rabbit anti-panAkt (11E7, Cell Signaling), and rabbit anti-phospho Akt Ser473 (D9E, Cell Signaling). Secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit (Cell Signaling). For chemiluminescent detection, the SuperSignal West Femto Substrate Kit was used according to the manufacturer's instructions (Thermo Fisher Scientific). Images were captured using a Carestream 4000M Pro Image Station and densitometry analysis was performed using ImageJ Software (W.S. Rasband, NIH, version 1.51).
4
Investigating Protein Expression
5
Chondrocyte Protein Immunoblot Analysis
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Individual chondrocyte pellet cultures were homogenized in 60 µL of Pierce IP Lysis Buffer (Thermo Scientific) with freshly added Halt Protease and Phosphatase Inhibitor (Thermo Scientific). Twenty μL of lysate was added to 7.5 µL of NuPAGE LDS Sample Buffer and 3 µl NuPAGE Reducing Agent, heated to 70°C for 10 minutes, and loaded onto a 4–20% NuPAGE Bis-Tris Mini Gel (Life Technologies). Following transfer to Invitrolon PVDF membranes (Life Technologies) using standard procedures, immunodetection was performed using the Western Breeze Chemiluminescent Immunodetection kit (Life Technologies). The following primary antibodies were used: mouse anti-SHP2 (M163; Abcam), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204; D13.14.4E; Cell Signaling), rabbit anti-ERK1/2 (137F5; Cell Signaling), and rabbit anti-phospho-AKT (Ser473;D9E; Cell Signaling). All primary antibodies were diluted to 1∶1000 and incubated overnight at 4°C.
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