Bronchial tissue samples were lysed in 1 mL of cell extraction buffer. Lysates were incubated in radio-immunoprecipitation assay buffer and kept at 4°C, for 2 h, followed by centrifugation at 12,000×g for 30 min at 4°C, and the supernatants collected. Total proteins were separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis on 8% gels, transferred onto polyvinylidene fluoride membranes, and blocked in TBST (plus 5% skim milk powder, pH 7.4, tris-buffered saline [TBS] with 0.24% Tween-20). Membranes were then incubated overnight at 4°C with the following primary antibodies: p-PI3K (Abcam, ab86714, 1:1,000), p-PKB (Abcam, ab81283, 1:5,000), p-PCNA (Abcam, ab92552, 1:3,000), p-AR (Abcam, ab3442, 1:1,000), and β-actin (Abcam, ab8226, 1:2,000). Blots were washed three-times for 15 min in TBST and treated with an horseradish peroxidase-conjugated polyclonal goat anti-rabbit antibody (Beyotime, A0208, 1:1,000) for 2 h at room temperature. Membranes were washed three-times for 10 min, and HRP substrate used to detect the HRP-secondary antibody bound to the proteins of interest, based on the manufacturer’s instructions. The p-PI3K, p-PKB, p-PCNA, and p-AR protein expression levels were standardized to those of β-actin.
Ab3442
Manufactured by Abcam
Sourced in United States
Ab3442 is an antibody designed for use in laboratory research. It is a polyclonal antibody produced in rabbits. The core function of this product is to serve as a detection tool for the target antigen in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (2 mentions)
Ab182136, by Abcam (2 mentions)
Mouse anti gfp antibody, by Merck Group (2 mentions)
Mouse m1 anti flag antibody, by Merck Group (2 mentions)
Saponin, by Merck Group (2 mentions)
Saponin, by Thermo Fisher Scientific (2 mentions)
Rabbit anti gfp antibody, by Thermo Fisher Scientific (2 mentions)
Ab86714, by Abcam (1 mentions)
Ab92552, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
A0208, by Beyotime (1 mentions)
Ab81283, by Abcam (1 mentions)
A7811, by Merck Group (1 mentions)
Ab8295, by Abcam (1 mentions)
Apc 017, by Alomone (1 mentions)
β actin, by Proteintech (1 mentions)
Fv1000, by Olympus (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Cy3 conjugated goat anti rabbit, by Jackson ImmunoResearch (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
12 protocols using ab3442
1
Protein Expression Analysis of Bronchial Tissue
2
Immunofluorescence Staining of Cardiac Markers
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Immunofluorescence staining was performed for detecting proteins. Images were collected using the fluorescence microscope Leica DMRE DFC3000G. The primary antibodies were monoclonal anti-α-actinin antibody (A7811, 1:200; Sigma-Aldrich, Merck KgaA, Darmstadt, Germany), anti-cardiac troponin T antibody (ab8295, 1:200; Abcam, Cambridge, UK), anti-Kv4.3 (KCND3) antibody (APC-017, 1:400; Alomone Labs, Jerusalem, Israel), and anti-beta 1 adrenergic receptor antibody (ab3442, 1:400; Abcam, Cambridge, UK).
3
Adipose Tissue Protein Analysis
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Briefly, 20 μg total lysates from tissues or cells were run on a 10% SDS-PAGE gel and immunoblotted with the primary antibodies (1:1000) to HuR (Millipore, #07-468), ATGL (abcam, ab109251), Perilipin-1 (abcam, ab172907), HSL(cst, 4107), Akt (cst, 4691), p-Akt (ser473, cst, 4060), ADRB1 (abcam, ab3442), ADRB2 (abcam, ab182136), ADRB3 (abclonal, A8607), β-actin (Proteintech, 66009-1), GAPDH (Proteintech, 60004-1). The intensity of bands was measured by using ImageJ. All experiments were repeated at least three times and mean values were derived. All the uncropped blots are included in the Source Data file.
4
Immunostaining of β1-Adrenergic Receptors
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The 150-μm-thick prefrontal cortex slices were fixed in 4% paraformaldehyde in PBS (at 4°C) for 6 h. The free-floating sections were blocked with 5% goat serum in PBS and incubated with the rabbit anti-β1-adrenergic receptor antibody (1:200, Abcam, catalog number: ab3442) overnight at 4°C. Unbound antibodies were washed out with PBS, and the Cy3-conjugated goat anti-rabbit (1:200, Jackson ImmunoResearch, catalog number: 111-165-144) secondary antibody was applied at room temperature for 2 h. Next, the sections were washed with PBS (four times for 5 min each), mounted in medium (Vectashield with DAPI, Vector Laboratories, catalog number H-1200) and coverslipped. The specificity of the applied primary antibodies (Abcam, catalog number: ab3442) was recently documented (Lv et al., 2016 (link); Tyurin-Kuzmin et al., 2016 (link); Huang et al., 2017 (link); Kim et al., 2017 (link); Mesquita et al., 2017 (link)). Immunofluorescence measurements were performed with a confocal laser scanning microscope (FV1000, Olympus, objectives 10× and 60×, image 1024 pixels × 1024 pixels).
5
Protein Expression Analysis in Cardiac Tissue
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Apical myocardia harvested from four to six hearts per treatment group were homogenized in a lysis buffer containing phosphatase and proteinase cocktail inhibitor, in a ratio of 100:1:1, respectively. Lysates of normalized concentrations treated with reducing agents were denatured at 100°C for 10 min and separated on SDS-PAGE gels. The transferred protein bands were blocked and immunoblotted overnight in the following primary antibodies: β1AR (ab3442; Abcam), β2AR (ab182136; Abcam), MEF2 (ab64644; Abcam), GRK5 (ab64943; Abcam) GATA4 (ab84593; Abcam), NFAT (ab25916; Abcam), AC5 (PAC-501AP; FabGennix), AC6 (PAC-601AP; FabGennix), AC7 (PAC-701AP; FabGennix), ANP (sc-515701; Santa Cruz Biotechnology), BNP (sc-271185; Santa Cruz Biotechnology), GRK2 (sc-13143; Santa Cruz Biotechnology), pNF-κB (3033T; Cell Signaling Technology), NF-κB (8242T; Cell Signaling Technology), Cleaved Caspase-3 (9661T; Cell Signaling Technology), Collagen Type I (14695-1-AP, Proteintech), Collagen Type III (13548-1-AP, Proteintech), and GAPDH (10494-1-AP; Proteintech). Immunoblots were performed in triplicates and normalized with their respective loading controls.
6
Western Blot Analysis of Autophagy Markers
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Total protein homogenates from the LV or lysate of NRCM underwent western blot analysis. The antibodies used in this study were follows: a monoclonal rabbit antibody to mouse Rubicon (#8465, Cell Signaling Technology), a monoclonal rabbit antibody to rat Rubicon (21444-1-AP, Proteintech), a monoclonal mouse antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (016-25523, FUJIFILM Wako Pure Chemical Corporation), a polyclonal rabbit antibody to p62 (PM045, MBL), a polyclonal rabbit antibody to LC3 (#2775, Cell Signaling Technology), a polyclonal rabbit antibody to 1 adrenergic receptor (ab3442, abcam), a monoclonal mouse antibody to phospholamban (ab2865, abcam), and a polyclonal rabbit antibody to phosphorylated phospholamban (07-052, Sigma Aldrich). The antibody to 1 adrenergic receptor was validated by western blotting analysis of HA-tagged mouse 1 adrenergic receptor (Supplementary Fig. S2). After incubation with secondary antibody, the blot was developed with ImmunoStar Zeta or ImmunoStar LD reagent (FUJIFILM Wako Pure Chemical Corporation). ImageQuantTL (version 7.0, Cytiva) was used for quantitative analysis to evaluate protein expression levels.
7
Cardiac Protein Expression Analysis
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Protein expressions of GRK2, βARKct, β1-AR, SERCA2a, and RYR2 were analyzed by the Western blot analysis. Myocardial tissues were homogenized in ice-cold lysis buffer, and crude supernatant was harvested by centrifugation at 20,000 g at 4°C for 20 min. Protein concentrations were determined by a BCA protein assay kit (Bioswamp, Wuhan, Hubei, China). The proteins were separated by electrophoresis through 4% to 12%
Tris/glycine gels, and transferred onto the nitrocellulose membrane. Membrane was blocked in 5% non-fat dried milk at room temperature for 1 h. The membrane was then incubated with anti-GRK2/3 (1:1,000 dilution; Upstate 05-465 Millipore-Sigma), anti-βARKct (1:1,000 dilution; sc-13143; Santa Cruz), anti-β1-AR (1:1,000 dilution; ab3442, Abcam), anti-type 2 ryanodine receptor (RyR2) (1:5,000 dilution; ab59225; Abcam), and anti-SERCA2 ATPase (1:5,000 dilution; ab2861; Abcam) primary antibodies, respectively, at 4°C overnight. The membrane was incubated with the horseradish peroxidase-linked secondary antibody (1:5,000 dilution). Protein bands were visualized using the enhanced chemiluminescence kit (Tiangen Biotech Co, Ltd, Beijing, China). Protein density was quantified using the Tanon-5200 detection system (Tanon Science & Technology Co, Ltd, Shanghai, China). GAPDH served as an internal reference.
Tris/glycine gels, and transferred onto the nitrocellulose membrane. Membrane was blocked in 5% non-fat dried milk at room temperature for 1 h. The membrane was then incubated with anti-GRK2/3 (1:1,000 dilution; Upstate 05-465 Millipore-Sigma), anti-βARKct (1:1,000 dilution; sc-13143; Santa Cruz), anti-β1-AR (1:1,000 dilution; ab3442, Abcam), anti-type 2 ryanodine receptor (RyR2) (1:5,000 dilution; ab59225; Abcam), and anti-SERCA2 ATPase (1:5,000 dilution; ab2861; Abcam) primary antibodies, respectively, at 4°C overnight. The membrane was incubated with the horseradish peroxidase-linked secondary antibody (1:5,000 dilution). Protein bands were visualized using the enhanced chemiluminescence kit (Tiangen Biotech Co, Ltd, Beijing, China). Protein density was quantified using the Tanon-5200 detection system (Tanon Science & Technology Co, Ltd, Shanghai, China). GAPDH served as an internal reference.
8
Antibody Characterization in Animal Studies
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Primary antibodies used in animal studies were listed as follows: Na+/K+-ATPase α 1 subunit (05–369, Millipore, CA, USA), glucose transporter 4 (Glut4, 2213, Cell Signaling Technology, MA, USA), insulin receptor substrate 1 (IRS1, sc-559, TX, Santa Cruz Biotechnology), phosphotyrosine (sc-7020, Santa Cruz Biotechnology, USA), phosphorylated AS160 (Tyr642) (4288, Cell Signaling Technology), total AS160 (07–741 Millipore), tumor necrosis factor (TNFα, ab6671, Abcam, MA, USA), ED-1 (MCA341, Serotec, Oxford, UK), angiotensinogen (AGT, 28101, IBL, Gunma, Japan), AGT (A6279, ABclonal, Wuhan, China), Ang II (T-4007, Peninsula Laboratories, CA, USA), dystrophin (ab129996, Abcam), adiponectin (ab22554, Abcam), CGRP (GTX16211, Gene Tex, CA, USA), tyrosine hydroxylase (TH, MAB318, Millipore), c-fos (ab209794, Abcam), Nox2 (ab129068, Abcam), p22phox (sc-271,968, Santa Cruz Biotechnology), α1-adrenergic receptor (AR) (ab3462, Abcam), α2-adrenergic AR (CA1003, Cell Application, CA, USA), β1-AR (ab3442, Abcam), β2-AR (ab182136, Abcam), β3-AR (sc-1473, Santa Cruz Biotechnology). Secondary antibodies were purchased from Cell Signaling Technology.
9
Cardiac Cell Immunofluorescence Protocol
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The cardiac tissues in different groups were prepared into frozen sections for immunofluorescence analysis. For cellular immunofluorescence, cells were washed three times with PBS. When the samples were prepared and fixed with 4% PFA for 10 min, 5% BSA was used to block for 1 h before the addition of primary antibodies. The primary antibodies were as follows: LCA (Fluorescein-LCA) (1:1,000, FL-1041-5, VectorLab, Burlingame, VT, United States) and β1AR (1:100, ab3442, Abcam, Cambridge, CB, United Kingdom). Secondary antibodies coupled with Alexa Fluor 594-conjugated Goat Anti-Rabbit (1:200, AS039, Abclonal, Wuhan, China) were used, and nuclei counterstaining was performed using DAPI (1:1,000, Beyotime, Beijing, China) for 10 min at room temperature for cell nuclei staining. Subsequently, samples were washed with 0.5% PBST three times for 3 min each time and sealed with an anti-fluorescence quencher (P0126, Beyotime, Beijing, China).
10
Western Blot Protein Analysis
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Western blots were executed with a standard protocol. In detail, membrane proteins were extracted using a membrane protein extraction kit (#P0033, Beyotime, China), and the protein concentration of the supernatant was determined by the BCA Protein Assay Kit (#CW0014S, CWBIO, China) [15] (link). Fifty micrograms of protein from each sample was loaded, separated by electrophoresis and transferred to a polyvinylidene uoride membrane (#ISEQ00010, Merck Millipore, USA). The membranes were blocked with a blocking buffer [5% nonfat milk in Tris-buffered saline (TBS) containing 50 mM Tris-HCl (pH 7.6) and 150 mM NaCl] for 1 h at RT and then incubated with the primary anti-β 1 ARs antibody at 4°C (1:500, ab3442; Abcam, USA). Subsequently, the membranes were washed three times with TBS-Tween-20 and incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h at RT. Protein bands were detected with an ECL chemiluminescence system (#KGP1127, KEYGEN BIOTECH, China), gauged by ImageJ software (National Institutes of Health, USA) and normalized to the internal control β-actin bands.
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