The collected Raw264.7 cells and bone surrounding soft tissues were homogenized and lysed in lysis buffer including RIPA and phenylmethanesulfonyl fluoride (PMSF; Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). The lysates (30 μg/lane) were separated on 10% gels and were transferred onto polyvinylidene difluoride membranes (Beyotime Institute of Biotechnology). The membranes were blocked with 5% (w/v) BSA in Tris-buffered saline with 0.1% (w/v) Tween 20 (TBST) and probed with primary antibodies: mouse anti-ABCA1 (1: 1000; Abcam Biotechnology), rabbit anti-ABCG1 (1: 2500; Abcam Biotechnology), rabbit anti-Cav-1 (1: 1500; Abcam Biotechnology), rabbit anti-TNF-α (1: 1000; Abcam Biotechnology), rabbit anti-IL-6 (1: 1000; Cell Signaling Technology), rabbit anti-IL-1β (1: 1,000; Abcam Biotechnology), and mouse anti-β-actin (1: 4000; Proteintech, Rosemont, USA) at 4°C overnight. After washing, the bound antibodies were detected using optimal horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit IgG (1: 10 000) and goat anti-mouse IgG (1: 10 000; Merck Millipore, Darmstadt, Germany) and were visualized using the enhanced chemiluminescence reagents (Bio-Rad Laboratories, Hercules, USA). The relative levels of the target proteins to that of control β-actin were analyzed densitometrically using the Quantity One software (version 4.0, Bio-Rad).
Rabbit anti cav 1
Manufactured by Abcam
Sourced in United States
Rabbit anti-Cav-1 is a primary antibody that detects the Caveolin-1 (Cav-1) protein. Caveolin-1 is a structural component of caveolae, which are invaginations of the plasma membrane involved in various cellular processes. This antibody can be used to detect and study the expression and localization of Caveolin-1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Hrp coupled secondary antibody, by Jackson ImmunoResearch (2 mentions)
Image lab 5, by Bio-Rad (2 mentions)
Mouse anti transferrin receptor, by Thermo Fisher Scientific (2 mentions)
Criterion xt 4 12 bis tris gel, by Bio-Rad (2 mentions)
Rabbit anti connexin 43 antibody, by Thermo Fisher Scientific (2 mentions)
Mouse anti phospho caveolin1 py14, by BD (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Rabbit anti β catenin, by Abcam (2 mentions)
Ecl plus, by GE Healthcare (2 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
Rabbit anti il 1β, by Abcam (1 mentions)
Rabbit anti tnf α, by Abcam (1 mentions)
Mouse anti β actin, by Proteintech (1 mentions)
Mouse anti abca1, by Abcam (1 mentions)
Rabbit anti abcg1, by Abcam (1 mentions)
Goat anti rabbit igg, by Merck Group (1 mentions)
Goat anti mouse igg, by Merck Group (1 mentions)
Rabbit anti il 6, by Cell Signaling Technology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Beyotime (1 mentions)
5 protocols using rabbit anti cav 1
1
Western Blot Analysis of Inflammatory Markers
2
Plasma Membrane Protein Analysis
3
Western Blot Analysis of Cellular Proteins
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Protein lysates were separated on 4–12% Bis-Tris gels and transferred to nitrocellulose membranes. The membranes were blocked for 60 min at room temperature in 5% milk in TBS-T (0.05% Tween-20, pH 7.4), washed with TBS-T and then incubated with primary antibody [rabbit anti-Cav-1 (Abcam, Cambridge, MA), mouse anti-Cav-1 (BD Technologies, Research Triangle Park, NC), rabbit anti-β-actin (Cell Signaling Technologies, Boston, MA), rabbit anti-MnSOD(Abcam), rabbit anti-AMPK pThr-172 (Abcam), rabbit anti-Nrf2(Santa Cruz), mouse anti-Keap1 (Abcam), rabbit anti-Cysteine Sulfenic Acid (EMD Millipore, Billerica, MA)] in TBS-T overnight at 4°C. Antibodies were diluted 1:1000 unless stated otherwise. After 3 washes, membranes were incubated with the infrared secondary antibody to anti-rabbit/mouse, 1:5,000 in TBS-T and incubated for 2 hours at room temperature. After 3 washes, protein signals were analyzed by Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
4
Sucrose Gradient Fractionation and Western Blot Analysis of Caveolin-1
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Cell lysates from 12 fractions of the sucrose gradient subcellular fractionation were used for Western blotting. Proteins were analyzed by SDS-PAGE in a 12% polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) blots which were blocked in 5% bovine serum albumin (BSA; Sigma) in Tris-buffered saline + 0.05% Tween 20 (TBST), and then incubated overnight at 4 °C with rabbit anti-CAV-1 (1:1000 in TBST+ 2.5% BSA; Abcam). The secondary antibody was Amersham ECL donkey anti-rabbit horseradish peroxidase (HRP)-linked IgG antibody (GE Healthcare UK Limited, Little Chalfont, UK). HRP activity was detected using Super Signal West Dura, Extended Duration Substrate (ThermoScientific™ Pierce™ Protein Biology) and the chemiluminescence reaction was visualized using a FOTO/Analyst FxCCD imaging system (Fotodyne Inc., Hartland, WI, USA).
The intensity of bands on Western blots was measured using grayscale images in Image J software (http://imagej.nih.gov/ij ) as described previously [32 (link)]. Intensities of CAV-1 bands were normalized to the total protein concentration from the same sample. Total protein concentration before sucrose gradient subcellular fractionation was determined by BCA assay (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s protocol.
The intensity of bands on Western blots was measured using grayscale images in Image J software (
5
Plasma Membrane Protein Analysis
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Three confluent 160-mm-diameter dishes of MBECs were harvested and plasma membranes were isolated as previously described (Yao et al., 2009). Protein concentrations were determined by Bio-Rad Dc-Protein assay (Bio-Rad). Samples were mixed with Bio-Rad XT sample buffer and reducing agent, run on Criterion™ XT 4–12% Bis-Tris Gels (Bio-Rad), and transferred onto PVDF membranes. Immunodetection was performed with (1:100) rabbit anti-connexin 43 antibody (Invitrogen), (1:500) mouse anti phospho-caveolin1 (pY14) (BD Transduction Laboratories), (1:5000) rabbit anti-β-catenin (Abcam), (1:1000) rabbit anti-Cav-1 (Abcam), or (1:1000) mouse-anti-transferrin receptor (Life Technology) followed by HRP-coupled secondary antibodies (Jackson ImmunoResearch), and developed with ECL-Plus (GE Healthcare). Protein bands were quantified by densitometry using Image Lab 5.1 (Bio-Rad) software. Plasma membrane connexin43 levels were normalized to plasma membrane transferrin receptor. This value was normalized to total β-catenin as a housekeeping reference protein.
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