Primary antibodies from Cell Signaling Technology (Danvers, MA, USA) were used to detect Caspase 3 (#9662; 1:1000/1:500 [full-length/cleaved]), Bad (#9239; 1:500), phospho-Bad (Ser112) (#5284; 1:500), Erk (#4695; 1:1000), phospho-Erk (#9101; 1:1000), Akt (#9272; 1:1000), phospho-Akt (#4060; 1:200), MK2 (#3042; 1:500), phospho-MK2 (Thr222) (#3316; 1:200), phospho-MK2 (Thr334) (#3007; 1:200), p38 (#9212; 1:1000), phospho-p38 (#4511; 1:2000), JNK (#9252; 1:1000), phospho-JNK (#9251; 1:500), c-Jun (#9165; 1:1000) and phospho-c-Jun (#3270; 1:1000). Primary antibodies against p53 (sc-47698; 1:1000), phospho-p53 (Ser392) (sc-51690; 1:500) and Vinculin (sc-73614; 1:1000) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Polyclonal antibodies against TCRV (1:1000) and JUNV (1:1000) NP (produced in guinea pigs [15 (link)]) were used to confirm viral infection. Fluorescently labeled secondary antibodies were purchased from LI-COR (IRDye 680RD Donkey anti-Mouse IgG, IRDye 680RD Donkey anti-Guinea Pig IgG, IRDye 800CW Donkey anti-Rabbit IgG) and used at a dilution of 1:15000. A goat anti-rabbit IgG HRP-linked secondary antibody (#7074; 1:5000, Cell Signaling Technology) was used for the detection of Bad.
Goat anti rabbit igg hrp linked secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Goat anti-rabbit IgG HRP-linked secondary antibody is a laboratory reagent used to detect and quantify target proteins in various immunoassays. It consists of goat-derived antibodies that specifically bind to rabbit immunoglobulin G (IgG) and are conjugated with the enzyme horseradish peroxidase (HRP).
Automatically generated - may contain errors
Lab products found in correlation
Sc 73614, by Santa Cruz Biotechnology (1 mentions)
Irdye 680rd donkey anti mouse igg, by LI COR (1 mentions)
Irdye 800cw donkey anti rabbit igg, by LI COR (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Phospho mk2 thr334, by Cell Signaling Technology (1 mentions)
Vinculin, by Santa Cruz Biotechnology (1 mentions)
Phospho bad ser112, by Cell Signaling Technology (1 mentions)
Phospho c jun, by Cell Signaling Technology (1 mentions)
C jun, by Cell Signaling Technology (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Rabbit anti histone h3, by Cell Signaling Technology (1 mentions)
Ecl detection system, by Cytiva (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Anti p62, by Cell Signaling Technology (1 mentions)
Image lab version 3, by Bio-Rad (1 mentions)
5 protocols using goat anti rabbit igg hrp linked secondary antibody
1
Immunoblot Analysis of Signaling Pathways
2
Western Blot Analysis of ERK2 and TCF21
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851FH-GFP and 851FH-TCF21 CAFs were lysed in RIPA lysis buffer supplemented with EDTA-free protease inhibitor cocktail tablets (Roche) and normalized for total protein amount. 35 µg protein from each sample was resolved in a 12% SDS-PAGE gel and transferred onto Immobilon-P membranes (Millipore) using a semidry transfer method (Bio-Rad). Blots were probed overnight at 4°C using a mouse-anti-human ERK2 antibody (Santa Cruz; 1:1,000) and a rabbit-anti-human TCF21 antibody (Sigma-Aldrich; 1:250), followed by a 45-min incubation at room temperature with a goat anti-mouse IgG HRP-linked secondary antibody (Invitrogen; 1:1,000), and goat-anti-rabbit IgG HRP-linked secondary antibody (Cell Signaling; 1:2,500). Proteins were detected using enhanced chemiluminescence reagent (ThermoFisher Scientific) and autoradiograph exposure (Sigma-Aldrich).
3
Western Blot Analysis of KLF4 and Histones
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Western blotting analysis was performed as described previously [44 (link)]. Briefly, cellular proteins were separated by 7% or 15% SDS-PAGE and transferred onto PVDF membranes (Whatman, Florham Park, NJ). The membranes were blocked with 5% (wt/vol) dry fat-free milk in TBS-T buffer (0.1% Tween) for 60 min at room temperature. Next, the membranes were incubated with rabbit anti-human KLF4 (4038, Cell Signal Technology, Danvers, MA), rabbit anti-histoneH3 (4499, Cell Signal Technology), or rabbit anti-β-actin (ACTB) antibodies (4967, Cell Signal Technology) in TBS-T buffer (5% BSA, 0.1% Tween) at 4 °C overnight. After washing with TBS-T buffer (0.1% Tween), the membranes were incubated with the goat anti-rabbit IgG HRP-linked secondary antibody (7074, Cell Signal Technology) at room temperature for 60 min. Proteins were visualized using an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia, Piscataway, NJ). Unprocessed images of western blots are shown in Additional file 1 : Fig. S10.
4
Autophagy Pathway Protein Expression Analysis
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The three key proteins [Beclin-1, sequestosome1(SQSTM1/p62), LC3B] expression of the autophagy pathway in the liver were detected by the western blotting. The antibodies used were anti-Beclin-1(No.3738, dilution1:1000, Cell Signaling Technology, Danvers, MA), anti-P62 (No.39749, dilution1:1000, Cell Signaling Technology, Danvers, MA), anti-LC3B (No.12741, dilution1:1000, Cell Signaling Technology, Danvers, MA) and anti-GAPDH (No.5174, dilution1:1000, Cell Signaling Technology, USA). All proteins were incubated for 1 h at room temperature with a goat anti-rabbit IgG HRP-linked secondary antibody (No.7074, Cell Signaling Technology, USA) and were visualized by a Chemi Doc XRS+ system (Bio-Rad, USA) and quantified in Image Lab version 3.0 (Bio-Rad, USA).
5
Detailed Reagents and Antibodies Protocol
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All cell culture reagents were obtained from Thermo Fisher Scientific, US unless otherwise stated. All chemicals were obtained from Sigma-Aldrich, UK unless otherwise stated. Ultrapure LPS (Invivogen, France), paxilline (Cayman Chemical Co., US), TATCN21 (sequence KRPPKLGQIGRSKRVVIEDDR) and scrambled control (sequence VKEPRIDGKPVRLRGQKSDRI) were obtained from Genscript (NJ, USA) [29 (link)]. Mouse monoclonal anti-mouse BKα antibody, clone L6/60 (EMD Millipore, US), Rabbit polyclonal anti-mouse lamin B1 antibody (Abcam plc., US), Rabbit monoclonal anti-mouse CREB antibody and Rabbit monoclonal anti-mouse Phospho (Ser133)-CREB antibody (Cell Signaling Technology, US), Mouse monoclonal anti-human COX IV antibody (Abcam plc. US), Rabbit monoclonal anti-human GADPH antibody (Cell Signalling Technology, US), Goat polyclonal anti-human Calnexin antibody (Sicgen, Portugal). All primary antibodies had been reported to detect the equivalent protein in mouse. Goat anti-rabbit IgG HRP-linked secondary antibody (Cell Signalling Technology, US), Rabbit anti-mouse IgG HRP-linked secondary antibody (Abcam plc., US), Rabbit anti-Goat IgG HRP-linked secondary antibody (R and D systems, US), F(ab)’ fragment affinity-purified unconjugated goat anti-mouse IgG (Thermo Fisher Scientific, US).
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