RNA was isolated from tissues using Trizol Reagent as per the manufacturer’s instructions (Life Technologies, Carlsbad, CA). cDNA was prepared as previously described (Harper et al., 2013 (link)). Polymerase chain reaction was carried out using Taq polymerase as per the manufacturer’s instructions and the following primers: Fgf21, forward: 5′-GGGGATTCAACACAGGAGAA-3′, reverse: 5′-AGGGCCTCAGGATCAAAGTGA-3′; and Glyceraldehyde 3-phosphate dehydrogenase, forward: 5′-ACGGCAAATTCAACGGCACAGTCA-3′, reverse: 5′-CATTGGGGGTAGGAACACGGAAGG-3′. Protein analyses were conducted as previously described (Girer et al., 2019 ) using the following antibodies: anti-HA C29F4 (Cell Signaling Technologies, Danvers, MA), anti-FGF21 Y-16 (clone sc-81946; Santa Cruz Biotechnology, Dallas, TX), or anti-actin (clone 13E5; Cell Signaling Technologies, Danvers, MA). Quantification of protein expression, normalized to actin, was performed using ImageJ software (Schneider et al., 2012 (link)).
Anti ha c29f4
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-HA (C29F4) is a rabbit monoclonal antibody that recognizes the influenza hemagglutinin (HA) epitope tag. This antibody is designed for use in various applications, such as immunoprecipitation, Western blotting, and immunohistochemistry, to detect and analyze HA-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2, by Merck Group (3 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (2 mentions)
Immobilon p pvdf membrane, by Merck Group (2 mentions)
Anti gapdh hrp conjugate a00192, by GenScript (2 mentions)
Amersham ecl, by Cytiva (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Anti flag f1804, by Merck Group (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Hek293ft cells, by American Type Culture Collection (1 mentions)
Anti flag agarose, by Merck Group (1 mentions)
Mw25000, by Polysciences (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Anti γ tubulin gtu 88, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Imaris 4, by Oxford Instruments (1 mentions)
Vectashield medium, by Vector Laboratories (1 mentions)
Anti s100, by Agilent Technologies (1 mentions)
20 protocols using anti ha c29f4
1
Quantifying FGF21 Expression in Tissues
2
Western Blot and Immunoprecipitation Antibodies
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Anti-HA (C29F4; 1:2000 dilution western blot) was obtained from Cell Signaling Technology. Anti-FLAG (F1804; 1:5000 dilution western blot; 1:1000 dilution immunoprecipitation) was obtained from Sigma-Aldrich.
3
Investigating p62 and ATF2 Interaction
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HEK293FT cells (ATCC, Manassas, VA, USA; #CRL-3216) or HEK293T cells (ATCC, Manassas, VA, USA; #CRL-1573) were cultured in 6-well plate and transfected with 1.5 μg Flag-p62 and 1.5 μg HA-ATF2 plasmid per well by polyethylenimine (linear, MW-25000, Polysciences, Hirschberg, Germany) for 24 h. Isoproterenol (10 μM) was added half an hour before the cells were collected. The protein lysate of 1500 μg was incubated with anti-Flag-agarose (Sigma-Aldrich, Munich, Germany) overnight for immunoprecipitation, together with 50 μg lysate as input to be loaded for western blotting detection of HA (anti-HA, Cell Signaling Technology) or Flag tag (anti-Flag). Antibodies were obtained from the following sources: anti-HA (C29F4) (Cell Signaling, Danvers, MA, USA; #3724S; dilution: 1:1000), anti-FLAG-M2 (Sigma-Aldrich, St. Louis, MO, USA; #F1804; dilution: 1:2000), anti-beta-actin (Cell Signaling, Danvers, MA, USA #4967S; dilution: 1:2000), anti-rabbit (Sigma-Aldrich, St. Louis, MO, USA; #A3687; dilution: 1:20,000), anti-mouse (Sigma-Aldrich, St. Louis, MO, USA; #A3562; dilution: 1:20,000).
4
Antibody Validation and Nocodazole Treatment
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Primary antibodies were as follows (with sources and references codes in parentheses): rabbit monoclonal anti-HA (C29F4; Cell Signaling), rabbit polyclonal anti-INPP5K (GTX32681; GeneTex), rabbit polyclonal anti-ARL6IP1 (GTX85516; GeneTex). Nocodazole was purchased from Sigma-Aldrich (product number M1404).
5
Immunofluorescence Staining of Cells
6
Western Blot Analysis of Protein Expression
7
Immunohistochemical Analysis of Tissues
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Tissues and tumor samples were fixed with 4% paraformaldehyde overnight and embedded in paraffin. Sections were stained with hematoxylin and eosin using standard protocol. For immunostaining, the antibodies used were anti-HA (C29F4) (Cell Signaling; dilution 1:100–500), anti-HA (Roche; dilution 1:50), anti-Ki67 (SP6) (Abcam; dilution 1:100–150), anti-BrdU (BU1/75 (ICR1)) (Abcam; dilution 1:100), anti-MBP (Abcam; dilution 1:100–200), anti-S100 (Dako; undiluted), anti-SOX10 (R&D; dilution 1:150), anti-GFP (4B10) (Cell signaling; dilution 1:150), anti-p53 (Ab240) (Abcam; dilution 1:250), and anti-p21 (HUGO291) (Abcam; dilution 1:100–500).
8
Immunoblotting and Immunostaining Antibodies
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Anti-FLAG monoclonal antibody (M2, Sigma), anti-HA monoclonal antibodies (3F10, Roche; 6E2, Cell Signaling Technology) and anti-actin polyclonal antibody (C-11, Santa Cruz Biotechnology) were used as primary antibodies for immunoblotting. Anti-HA (C29F4, Cell Signaling Technology) was used as primary antibody for immunostaining. Horseradish peroxidase-linked anti-mouse (GE Healthcare), anti-rat (GE Healthcare) and anti-goat (Santa Cruz Biotechnology) antibodies were used as secondary antibodies in immunoblotting. Alexa Fluor 546 anti-rabbit antibody (Invitrogen) was used in immunostaining as secondary antibody.
9
Immunofluorescence Protocol for HA and PLN
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Cells were fixed with 4% PFA in PBS, permeabilised with ice-cold acetone and subsequently blocked in 1.1% BSA in PBS overnight. Cells were subsequently stained using anti-HA (C29F4, Cell Signalling Technology, USA) and anti-PLN (2D12, Thermo Fisher, USA) antibodies at a 1:400 dilution in blocking buffer, overnight. Cells were washed with blocking buffer and incubated with fluorescently labelled secondary antibodies and nuclei were stained with Gold Anti-fade Reagent with DAPI (Invitrogen, USA). Imaging was performed using a Zeiss axio observer fluorescence microscope (Zeiss, Germany). The use of the anti-PLN antibody 2D12 for immunofluorescence studies was validated using adult murine heart tissue (Supplementary Fig. 6 ).
10
Western Blot Analysis of Protein Expression
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Whole-cell lysates were prepared by boiling cells in Laemmli Sample Buffer (Bio-Rad) supplemented with 100 mM DTT for 10 min at a final concentration of 107 cells per milliliter. A total of 105 cells or 20 mg protein was resolved by SDS-PAGE and transferred to Immobilon-P PVDF membranes (Millipore). Membranes were blocked in 5% milk in TBS-T (25 mM Tris-HCl [pH 7.4], 0.13 M NaCl, 2.7 mM KCl). Primary antibodies (anti-HA [C29F4; Cell Signaling Technologies], anti-DPEAAE [PA1-1748A; Thermo]) were diluted in 5% milk-TBS-T, and membranes were incubated overnight at 4°C. Secondary Ab–HRP conjugate, as well as anti-GAPDH–HRP conjugate (A00192; GenScript), incubations were carried out for 1 h at room temperature. Signal detection was achieved using Amersham ECL.
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