Western blot was carried to evaluate protein expression levels as described previously [33] (link). The transferred proteins were detected with primary antibodies as follows: p110β antibody (#3011), p110α antibody (#4249), p110γ antibody (#5405), akt antibody (#4691), and phospho akt antibody (#4060) were purchased from Cell Signaling Technology. The β-actin antibody (ab8227) was from Abcam. We detected the immunoblot signals using ChemiDoc XRS (Bio-Rad Laboratories).
P110α antibody
Manufactured by Cell Signaling Technology
The P110α antibody is a tool used in research laboratories. It is an antibody that specifically binds to the P110α protein, which is a subunit of the phosphoinositide 3-kinase (PI3K) enzyme complex. The P110α antibody can be used to detect and quantify the presence of the P110α protein in biological samples.
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Lab products found in correlation
Pi3k kinase activity elisa pico kit, by Echelon Biosciences (2 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Akt antibody, by Cell Signaling Technology (1 mentions)
Phospho akt antibody, by Cell Signaling Technology (1 mentions)
α tubulin antibody t6074, by Merck Group (1 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Irdye 680rd donkey anti mouse igg, by LI COR (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Tgx 221, by Selleck Chemicals (1 mentions)
Anti puromycin antibody, by Developmental Studies Hybridoma Bank (1 mentions)
6 protocols using p110α antibody
1
Evaluating Cellular Signaling Protein Levels
2
Western Blotting Antibodies and Inhibitors
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The phospho-S6 antibody (Ser235/236, #2211), S6 antibody (#2317), and p110α antibody (#4255) were obtained from Cell Signaling Technology. p110δ antibodies (#sc-7176, mab2687) were obtained from Santa Cruz Biotechnology and R&D Biosciences, respectively. The p110β antibody (#09-482) was obtained from EMD Millipore, the α-tubulin antibody (T6074) was obtained from Sigma-Aldrich, and the anti-Puromycin antibody was purchased from the Developmental Studies Hybridoma Bank (University of Iowa). Secondary antibodies for Western blotting were obtained from VWR (horseradish peroxidase-coupled anti-mouse and anti-rabbit antibodies for ECL) or from Li-COR Biosciences (IRDye 680 RD donkey anti-mouse IgG and IRDye 800CW goat anti-rabbit IgG for infrared fluorescent Western blotting). Puromycin was purchased from Life Technologies, the p110δ inhibitor IC87114 was purchased from EMD Millipore, and the p110β inhibitor TGX-221 was purchased from Selleck Chemicals.
3
Quantifying Protein Expression with p110α Antibody
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Protein expression was quantified using p110α antibody (1:500; Cell Signaling, 4249), as described previously (Sferruzzi-Perri et al., 2011 (link)).
4
Hepatic Endotoxin and PI3K Activity Assays
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Liver tissues were homogenized in an ice-cold water bath and centrifuged for 10 min at 2500 g to remove connective tissue. The Limulus Amebocyte Lysate Endotoxin Detection Assay kit (Lonza, Walkersville, MD) was used to determine hepatic endotoxin levels following the steps recommended by the manufacturer. For the measurement of Pi3K enzymatic activity, Hepa1-6 cells were treated with 5 µM C37 or C646 inhibitor for 4 h. P110α antibody (Cell Signaling) was used to pull down Pi3K, and its enzymatic activity was measured using the PI3K-Kinase Activity ELISA: Pico kit (Echelon Biosciences).
5
Hepatocyte PI3K Kinase Activity Assay
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Twenty-four hours after the seeding, primary hepatocytes from ob/ob mice were subjected to serum starvation for 2 h, then treated with 20 μM of C37 or C646 for 3 h. P110α antibody (Cell signaling) was used to pull down PI3K, and its enzymatic activity was measured using the PI3K-Kinase Activity ELISA: Pico kit (Echelon Biosciences).
6
Immunoprecipitation and Ubiquitin Pulldown
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Cells were lysed in 20 mM TrisHCL pH 7.5, 137 mM NaCl, 1 mM EDTA, 1% NP40, 10% glycerol plus protease and phosphatase inhibitors. For p110α immunoprecipitation, lysates were incubated with p110α antibody (Cell Signaling, 4249) overnight. 50 µL of proteinA agarose beads were added to each sample and incubated additional 2 hours. For ubiquitinated protein pulldown experiment, cells were lysed in lysis buffer containing 200 µg/mL TUBE1 (Lifesensors UM101).
Lysates were isolated and added 50 µL of glutathione agarose beads (Sigma, G4705). The samples were incubated overnight and captured ubiquitinated protein was eluted in SDS reducing sample buffer.
Lysates were isolated and added 50 µL of glutathione agarose beads (Sigma, G4705). The samples were incubated overnight and captured ubiquitinated protein was eluted in SDS reducing sample buffer.
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