Mouse islets were preincubated for 1 h in 1 mmol/L glucose Krebs‐Ringer Bicarbonate (KRB) and subsequently treated for 20 min with P2Y1 agonists, ATP (10 μmol/L) and MRS2365 (100 nmol/L; EC50 0.4 nmol), or 30‐mmol KCl ± P2Y1 antagonist, MRS2500 (1 μmol/L; EC50 0.78 nmol). Following treatment, cells were lysed in buffer containing (in mmol/L): 20 Tris‐HCl (pH = 7.5); 150 NaCl, 1 EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1 EDTA, 1 b‐glycerophosphate, 25 N‐ethylmaleimide, 1% Triton X‐100, and 1X protease inhibitor cocktail. Protein lysates were separated using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), transferred to nitrocellulose membranes, incubated overnight at 4°C with primary antibodies (phospho‐PKD/PKCmu (serine 916) Rabbit A [Cell signalling] and anti‐β‐actin; sc‐4778 [Santa Cruz Biotechnology]), and visualized with horseradish peroxidase‐labeled anti‐rabbit IgG as secondary antibodies (Amersham, Baie d’Urfe, PQ). Images were acquired using a ChemiDoc MP System (Bio‐Rad) and analyzed using the densitometry analysis function in Image Lab Software 5.2.1 (Bio‐Rad) and normalized to actin as a loading control and expressed as a fold increase from baseline.
Anti β actin sc 4778
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-β-actin; sc-4778 is a mouse monoclonal antibody that recognizes the beta-actin protein. Beta-actin is a ubiquitously expressed cytoskeletal protein that plays a fundamental role in various cellular processes. This antibody can be used for the detection of beta-actin in a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software 5, by Bio-Rad (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Horseradish peroxidase labeled anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Mini protean tgx stain free gel, by Bio-Rad (1 mentions)
Na934v, by GE Healthcare (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Anti pka c α, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase, by Cell Signaling Technology (1 mentions)
Anti lamin a c sc 376248, by Santa Cruz Biotechnology (1 mentions)
Anti histone h3 ab1791, by Abcam (1 mentions)
3 protocols using anti β actin sc 4778
1
P2Y1 Receptor Modulation of Islet Signaling
2
Insulin Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
INS 832/13 cells were grown to 100% confluence in 6-well plates. Cells were treated with wortmannin (100 nM) or equivolume DMSO (0.1%) in KRB at the beginning of the 2.8 mM glucose 2-hr pre-incubation at 37 °C; these compounds were present for the duration of the experiment. Cells were treated with insulin (200 nM) or forskolin (1 μM) for 15 min in 16.7 mM KRB. Cells were then washed once with PBS, and then lysed using a standard RIPA lysis buffer supplemented with phosphodiesterase and protease inhibitor cocktails (Millipore, Etobicoke, ON).
Cell lysates were subjected to SDS-PAGE on Mini-PROTEAN TGX Stain-Free gels (Bio-Rad, Mississauga, ON) transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA), and probed with primary antibodies: anti-phospho-(Ser/Thr) AKT substrate; #9611, anti-phospho-(Ser/Thr) PKA substrate; #9621, anti-AKT; #9272, anti-PKA C-α; #4782 (Cell Signaling Technology, Beverly, MA) and anti-β-actin; sc-4778 (Santa Cruz Biotechnology). Detection was with peroxidase-conjugated secondary anti-rabbit (NA934V) and anti-mouse antibodies (NA931V) (GE Healthcare), and visualization by chemiluminescence with ECL-Plus (GE Healthcare). Images were acquired using a ChemiDoc MP System (Bio-Rad) and analyzed using Image Lab Software 5.2.1 (Bio-Rad).
Cell lysates were subjected to SDS-PAGE on Mini-PROTEAN TGX Stain-Free gels (Bio-Rad, Mississauga, ON) transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA), and probed with primary antibodies: anti-phospho-(Ser/Thr) AKT substrate; #9611, anti-phospho-(Ser/Thr) PKA substrate; #9621, anti-AKT; #9272, anti-PKA C-α; #4782 (Cell Signaling Technology, Beverly, MA) and anti-β-actin; sc-4778 (Santa Cruz Biotechnology). Detection was with peroxidase-conjugated secondary anti-rabbit (NA934V) and anti-mouse antibodies (NA931V) (GE Healthcare), and visualization by chemiluminescence with ECL-Plus (GE Healthcare). Images were acquired using a ChemiDoc MP System (Bio-Rad) and analyzed using Image Lab Software 5.2.1 (Bio-Rad).
3
Western Blot Analysis of Endometrial Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Endometrial cancer cells were lysed in a RIPA buffer (50 mM Tris HCl pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM PMSF). Concentrations of protein were determined using the Lowry method. Proteins of the cell lysates were resolved by 8% SDS-PAGE and transferred to Immobilon P membranes. The blots were incubated for two hours at room temperature with the following primary antibodies: anti-OGT (#5368) (diluted 1:2000, Cell Signaling Technology, Danvers, MA, USA), anti-lamin A/C (sc-376248) (diluted 1:2000; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Histone H3 (ab1791) (diluted 1:2500; Abcam), and anti-β-actin (sc-4778) (diluted 1:5000; Santa Cruz Biotechnology, Dallas, TX, USA). After washing with TBST (Tris buffered saline with Tween-20), immunoblots were incubated 1h at room temperature with goat anti-mouse or anti-rabbit secondary antibodies conjugated with horseradish peroxidase (diluted 1:5000, Cell Signaling Technology, Danvers, MA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!