N2a cells were washed with PBS and lysed in a modified radioimmunoprecipitation assay (RIPA) lysis buffer (#9806, Cell Signaling Technology) with 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein concentrations were determined with a BCA Protein Assay kit (#23227, Thermo). An equal amount of protein lysate was separated by 8% or 10% SDS-polyacrylamide gels and transferred to PVDF membranes (3,010,040,001, Roche). Membranes were blocked in TBST containing 5% non-fat dried milk and incubated with primary antibodies overnight at 4 °C. The membranes were washed with TBST and incubated with secondary antibody (1:2000 dilutions in 5% non-fat dried milk) for 2 h at room temperature (RT). Bound antibodies were visualized by chemiluminescent HRP substrate (#32209, Thermo). The mean densities of protein bands were measured by Image J software. The primary antibodies used are as follows: anti-rabies Virus (5B12) (NB110–7542, Novus) (1:1000), GAPDH (1A6) mAb (MB001, Bioworld) (1:5000), Clathrin Heavy Chain (P1663) Antibody (#2410, Cell Signaling Technology) (1:500), Caveolin-1 Antibody (#3238, Cell Signaling Technology) (1:500).
Caveolin 1 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Caveolin-1 antibody is a laboratory reagent used to detect and quantify the presence of the Caveolin-1 protein in biological samples. Caveolin-1 is a key structural component of caveolae, which are invaginations in the plasma membrane that play a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Chemiluminescent hrp substrate, by Thermo Fisher Scientific (1 mentions)
P1663 antibody, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Na k atpase antibody, by Cell Signaling Technology (1 mentions)
Pkcα antibody, by Cell Signaling Technology (1 mentions)
Mouse anti gapdh antibody, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Silver enhancement kit, by Merck Group (1 mentions)
4 protocols using caveolin 1 antibody
1
Western Blot Analysis of N2a Cells
2
Immunoprecipitation of Caveolin-1 and IGF-1R
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Briefly, 500 μg of protein lysate from ID8EV, ID8hPON2, scrambled siRNA-treated, and PON2 siRNA-treated cells were incubated with Caveolin-1 antibody (# 3267S, Cell Signaling Technology, Danvers, MA) for overnight at 4 °C. Subsequently, antigen–antibody complexes were incubated with Protein A agarose beads for 3 h at 4 °C. Following three washes, pellets were resuspended with 3× SDS sample Buffer. Negative control was performed without antibody. IGF-1R was detected by western blotting. Caveolin-1 and IGF-1R were also detected from total lysate (50 μg protein) as input controls.
3
Western Blot Analysis of Liver Proteins
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Proteins were extracted from liver tissues and HepG2 cells using a cell membrane protein and plasma protein extraction kit (Beyotime, P0033), and the following antibodies were used for Western blot (WB) analyses: BSEP polyclonal antibody (PA5‐78690, Invitrogen, Carlsbad, CA, USA); caveolin‐1 antibody (Cell Signaling Technology, Inc., Danvers, MA, USA); anti‐Hax‐1 antibody (Ab137613, Abcam plc., Cambridge, UK); PKCα antibody (Cell Signaling Technology, Inc.); phospho‐PKCα/β II (Thr638/641) antibody (Cell Signaling Technology, Inc.); mouse anti‐GAPDH antibody (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China); Na,K‐ATPase antibody (Cell Signaling Technology, Inc.); and mouse anti‐β actin antibody (Zhongshan Golden Bridge Biotechnology Co., Ltd.). Protein expression was normalized to β‐actin, GAPDH and Na+, K+‐ATPase. Densitometry analyses were performed using the ImageJ software.
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4
Caveolin-1 Immunogold Labeling in Mouse Brain
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Caveolin-1 immuno-EM labeling was performed as described previously (Andreone et al., 2017) . Mice were anesthetized and perfused through the heart with 30 mL PBS, followed by first 150 mL of a 5% glutaraldehyde/4% PFA/0.1 mM phosphate buffer fixative solution, then a 4% PFA/0.1 mM phosphate buffer fixative solution. Brains were dissected and post-fixed in 4% PFA/PBS for 30 min at 4 C. Coronal vibratome free-floating sections of 50 mm were collected and immersed in 0.1% sodium borohydride/PBS for 20 min at room temperature, blocked with 10% goat serum/PBST (0.01% Triton X-100), and incubated with Caveolin-1 antibody (1:100, Cell Signaling Technologies. 3267) overnight at room temperature. Sections were then incubated with gold-labeled goat antirabbit IgG (1:50, Nanoprobes. 2003) overnight at room temperature, washed with PBS and sodium acetate, and silver-enhanced using Silver Enhancement kit (Sigma, SE100) prior to processing for TEM. Data were collected from 4 mice per group (8 capillary cross-sections per mouse) and analyzed in a double-blind fashion. Randomization procedures are not applicable to these experiments. G-power software was used for sample size estimation. No data were excluded.
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