Cell lysis and immunoblotting were essentially performed as described previously [8 (link),9 (link),10 (link)] with minor modifications. Proteins were fractionated by polyacrylamide gel electrophoresis on TGX Stain-Free FastCast gels (BioRad, Munich, Germany). Following blotting and antibody treatment, chemoluminescent detection of proteins was carried with a ChemiDoc XRS+ System with Image Lab Software (BioRad) with Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to either the total amount of protein in the same lane (when using the TGX Stain-Free FastCast gels), or to bands for the housekeeping gene GAPDH. Significant differences (p < 0.05)-calculated with the unpaired two-tailed Student’s t test–denoted by bars above the respective cell lines. The antibodies used were Rac1b (#09-271, Merck Millipore, Darmstadt, Germany), E-cadherin (#610181) and Rac1 (#610650) (BD Transduction Laboratories, Heidelberg, Germany), GAPDH (14C10, #2118, Cell Signaling Technology, Frankfurt am Main, Germany), Vimentin (clone V9, #V6630, Sigma-Aldrich, Steinheim, Germany), and HA (clone 12CA5, Roche Diagnostics, Mannheim, Germany).
Ha clone 12ca5
Manufactured by Roche
Sourced in Germany
HA (clone 12CA5) is a mouse monoclonal antibody that recognizes the hemagglutinin (HA) epitope tag. It can be used for the detection and purification of proteins fused to the HA tag.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant 5, by GE Healthcare (2 mentions)
Nupage system, by Thermo Fisher Scientific (2 mentions)
Chemidoc xrs system with image lab software, by Bio-Rad (1 mentions)
Tgx stain free fastcast gels, by Bio-Rad (1 mentions)
Rac1b, by Merck Group (1 mentions)
E cadherin, by BD (1 mentions)
Vimentin clone v9, by Merck Group (1 mentions)
Amersham ecl prime detection reagent, by GE Healthcare (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ccar2, by Fortis Life Sciences (1 mentions)
Flag clone m2, by Merck Group (1 mentions)
Myc clone 9e10, by Merck Group (1 mentions)
P53 ac k382, by Cell Signaling Technology (1 mentions)
Sepharose beads, by Merck Group (1 mentions)
Cleaved parp 1, by Cell Signaling Technology (1 mentions)
Phospho chk2 t68, by Cell Signaling Technology (1 mentions)
Phos tag, by Fujifilm (1 mentions)
Sirt1, by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
4 protocols using ha clone 12ca5
1
Cell Lysis and Immunoblotting Protocol
2
Western Blot and Immunoprecipitation Analyses
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The NuPAGE system (Life Technologies) was used for western blot analyses and densitometric evaluations were performed with the ImageQuant 5.2 software (Molecular Dynamics). Quantification of protein levels were normalized to loading control and for phosphorylated proteins to total protein. Antibodies used in this study were: CCAR2 (Bethyl Laboratories or Cell Signaling Technology); phospho-Chk2-T68, phospho-Chk2-T387, Cleaved Caspase-9, KAP1, phospho-KAP1-S824, SIRT1, phospho-p53-S20 (Cell Signaling Technology); phospho-KAP1 S473 (Biolegend); 53BP1 (Novus), γH2AX and H3K9me3 (Upstate); FLAG (clone M2) and β-Actin (Sigma); HA (clone 12CA5, Roche); HP1β (Epigentek); phospho-ATM-S1981 (R&D); ATM (Epitomics); p53 (Santa Cruz, DO-7). Chk2 antibody (clone 44D4/21) was previously described [45 (link)] and used for IP. For western blot Chk2 antibody from MBL Intl Corp (DCS-270 and DCS-273) was used. IP experiments were carried out as described [46 ] except for the interaction between HP1α and KAP1 that was assayed after cell lysates sonication and co-immunoprecipitations of 53BP1 and H3K9me3 that were performed as reported [20 (link)].
3
Immunoprecipitation and Western Blot Protocol
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Transfected cells were lysed in Nonidet P-40-desoxycholate buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% Na desoxycholate, 0.5 mM Tris(2-carboxyethyl)phosphine, and 0.5 mM Pefabloc). Immunoprecipitation were carried out by addition of antibodies diluted 1:250 and incubation at 4 °C for 1h30. Protein A sepharose beads were added and incubated with the mix for 1 h. Beads were collected by centrifugation and washed three times with Nonidet P-40-desoxycholate buffer. For experience in Supplementary Fig. 1 protein G magnetic beads were used. Proteins were eluted in 2× SDS sample buffer at 80 °C for 10 min. After protein gel electrophoresis and transfer to PVDF membrane, detection was carried out by incubation with primary antibodies diluted 1:1000 or as indicated by the manufacturer and revelation was performed by chemiluminescence using ECL, ECL + or ECL Prime kits. Following antibodies were purchased: FLAG (clone M2, Sigma), MYC (clone 9E10, Sigma), HA (clone 12CA5, Roche), SMAD3 (NB600-1258), SMAD2/3 (566412, Calbiochem) and TAL1 (BTL73, Millipore).
4
Profiling Protein Modifications via Western Blot
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Western blot (WB) analyses were performed with the NuPAGE system (Invitrogen) and densitometric evaluations were done with the ImageQuant 5.2 software (Molecular Dynamics). REGγ phosphorylation analyses were performed using Phos-Tag (Wako) at 30 μM in a 10% acrylamide gel (13 (link)). The antibodies used were: DBC1 (Bethyl Laboratories, A300–434A or Cell Signaling Technology, clone 3G4); p53 (Santa Cruz, DO-7); p53-Ac-K382, phospho-Chk2-T68, PUMA, Cleaved PARP-1 and Cleaved Caspase-3 (Cell Signaling Technology); FLAG (clone M2), β-Actin and SIRT1 (Sigma); HA (clone 12CA5, Roche); REGγ (Transduction Laboratories). Chk2 antibody (clone 44D4/21) was previously described (33 (link)) or purchased from MBL Intl Corp (DCS-270 and DCS-273). Immunoprecipitation (IP) experiments were carried out as described (12 (link)) using cell lysates prepared in ELB buffer (150 mM NaCl, 50 mM Hepes pH 7.5, 5 mM EDTA, 0.5% NP40). After preclearing with protein-G (for mouse antibodies) or protein-A (for rabbit antibodies) coupled sepharose beads (Sigma), proteins of interest were subjected to IP with specific antibodies and analyzed by WB.
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