An amount of 0.3 × 106 cells were seeded in 6-well plates and incubated overnight. Cells were treated for 24 h with 1/2× and 1× IC50 concentrations. Whole-cell lysates were prepared using RIPA buffer supplemented with protease and phosphatase inhibitors. Lysates were sonicated and centrifuged at 20,000× g at 4 °C to remove insoluble material. Protein concentration was determined using a Micro BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and equal amounts of protein were resolved using NuPAGE Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA, USA). Western blot analysis was performed using DCK (GeneTex, Irvine, CA, USA: GTX107636; 1:2000), RRM2 (GeneTex: GTX103193; 1:2000), TK1 (GeneTex: GTX62133; 1:2000), RRM1 (Cell signaling, Beverly, MA, USA: 8637S; 1:3000), TYMS (Cell signaling: 9045P; 1:3000), DTYMK (Protein Tech, Rosemont, IL, USA: 15360-1-AP; 1:3000), DHFR (Protein Tech:15194-1-AP; 1:3000), IMPDH1 (Protein Tech: 22092-1-AP; 1:3000), GMPS (Santa Cruz, Dallas, TX, USA: sc-376163; 1:3000), p53 (Santa Cruz: sc-126; 1:3000), ETS2 (Santa Cruz: sc-365666; 1:200), γH2AX (Bethyl: A300-081A; 1:2000), and actin HRP (Sigma, St. Louis, MO, USA: A3854; 1:40,000) antibodies.
Actin hrp
Manufactured by Merck Group
Sourced in United States
Actin-HRP is a conjugate of actin, a key structural protein in cells, and horseradish peroxidase (HRP), an enzyme commonly used as a labeling agent. This product is designed for detection and visualization applications in cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Flag m2, by Merck Group (2 mentions)
Ecl solution, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab113692, by Abcam (2 mentions)
Hif 2α nb100 122, by Novus Biologicals (2 mentions)
Hif 1α, by BD (2 mentions)
Hpa001849, by Merck Group (2 mentions)
Riok3, by Abnova (2 mentions)
Ca9 clone m75, by Abcam (2 mentions)
Tmod3, by Merck Group (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Igf1r, by Abcam (2 mentions)
Smo ab72130, by Abcam (2 mentions)
Transferrin receptor tf r, by Santa Cruz Biotechnology (2 mentions)
Caveolin 1, by Santa Cruz Biotechnology (2 mentions)
Protease and phosphatase inhibitors cocktail, by Merck Group (2 mentions)
Psma7, by Thermo Fisher Scientific (2 mentions)
Uchl1, by Cell Signaling Technology (2 mentions)
Uchl1, by R&D Systems (2 mentions)
20 protocols using actin hrp
1
Protein Expression Analysis after Treatment
2
Comprehensive Immunofluorescence and Western Blot Protocol
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We used the antibodies in parentheses to the following antigens: ATG9A (Abcam, catalog #108338), FLAG M2 (Sigma, F1804), PAK3 (Abnova, PAB2300), SPTLC2 (Abcam, ab23696), GFP-HRP (MACS, 130091833), actin-HRP (Sigma, A3854), beclin 1 (Cell Signaling, 3738), ATG7 (Cell Signaling, 8558), ATG5 (Cell Signaling, 12994), ATG12 (Cell Signaling, 4180), ATG16 (Cell Signaling, 8089), LC3 (Cell Signaling, 3868), LAMP-1 (Cell Signaling, 9091), SNAP29 (Abcam, 138500), gp41 (NIH AIDS Reagent Program, 2F5), TGN46 (Bio-Rad, AHP500G), anti-HIV immunoglobulin (NIH AIDS Reagent Program, HIV-Ig), Nef (NIH AIDS Reagent Program, 2949), α-tubulin (Sigma, T5168), VSV-G (Sigma, V5507), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen, A21206), Alexa Fluor 488- conjugated donkey anti-mouse IgG (Invitrogen, A21202), Alexa Fluor 555-conjugated donkey antirabbit IgG (Invitrogen, A31572), Alexa Fluor 555-conjugated donkey anti-mouse IgG (Invitrogen, A31570), Alexa Fluor 555-conjugated donkey anti-sheep IgG (Invitrogen, A21436), HRP-conjugated donkey anti-rabbit IgG (GE Healthcare, NA934V), and HRP-conjugated sheep anti-mouse IgG (GE Healthcare, NXA931).
3
Phosphorylation Signaling in Mesangial Cells
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After 24 h of treatment, mesangial cells were lysed with lysis buffer (1 M Tris (pH = 7.4), 1.5 M NaCl, 1% Triton-X, 10% Glycerol, 50 mM EDTA (pH = 8), 0.1 M Sodium vanadate, 0.1 M PMSF, 0.1% protease inhibitor cocktail (Calbiochem) Samples were boiled for 5 minutes, electrophoresed on 8% or 12% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with specific Antibodies (p-AKT, p-GSK3β, p-FoxO3a, PTEN, p-(rp)S6 and p-mTOR, (Cell Signaling) actin HRP (Sigma). Blots were developed using horseradish peroxidase-conjugated secondary Abs and the ECL detection system (Thermo scientific, Pierce research protein products (Rockford, IL, USA)).
4
Western Blot Analysis of Signaling Proteins
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Cells were washed with PBS and lysed in extraction buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF and protease inhibitor cocktails]. Cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore corporation, Billerica, MA, USA). Membranes were blocked with 5% skim milk in TBS-T [10 mM Tri-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20] and immunoblotted with antibodies against c-Fos (Santa Cruz Biotechnology, Dallas, TX, USA), NFATc1 (Santa Cruz Biotechnology), Phospho-STAT5 (Cell Signaling Technology), STAT5A (Cell Signaling Technology, Beverly, MA), IκB (Cell Signaling Technology), Flag-HRP (Sigma-Aldrich), Actin-HRP (Sigma-Aldrich), mouse-IgG-HRP (Abcam, Cambridge, UK) and rabbit-IgG-HRP (Abcam). Signals were detected with ECL solution (Millipore corporation) and analyzed using a LAS3000 luminescent image analyzer (GE Healthcare, Piscataway, NJ, USA).
5
Pancreas Tissue Protein Extraction and Western Blot
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Pancreas tissue was snap frozen in liquid nitrogen was lysed on ice with RIPA buffer containing HALT Protease and Phosphatase inhibitor cocktail. Primary antibody incubation was performed overnight at 4°C in Tris-buffered saline containing 5% milk and 0.05% Tween-20. The following antibodies were used: GFP (Cell Signaling Technology 2956), Arid1a (Cell Signaling Technology 12354), Cpa1 (R and D AF2765), Amylase (Sigma A8273) and Actin-HRP (Sigma A3854). Blots were developed with SuperSignal West Femto substrate (ThermoFisher).
6
Western Blot Analysis of Hypoxia Markers
7
Protein Purification and Antibody Preparation
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A list of plasmids is provided in Table S1 . Plasmids were prepared by standard methods. Antibodies against the following proteins were purchased: UBQLN1/2 (Sigma, clone 5F5), UBQLN4 (Abcam ab106443), L9 and Tom20 (Santa Cruz Biotech., T-17 and FL-145), HA (Covance, clone 16B12), Rpt5 (Abcam ab22635), α7 (Enzo Life Sciences PW8110), FLAG (Sigma, clone M2), Hsc/Hsp70 (AssayDesigns, SPA-822), Hsp60 (Abcam ab46798), ClpP (Abcam ab124822), Actin-HRP (Sigma A3854), OxPhos cocktail (Abcam ab110411). FLAG-M2 Affinity resin was from Sigma, and GFP-trap from Chromotek. Antibodies to Bag6, TRC40, SGTA, TRAPα, GFP, and RFP have been described (Fons et al., 2003 (link), Mariappan et al., 2010 (link), Hessa et al., 2011 (link)). Anti-Myc was clone 9E10. Anti-HA used for IPs and blots (e.g., Figure 2 A) was raised in rabbits against the KLH-HA peptide conjugate. Recombinant proteins were either purchased (His6-Ubiquitin, E1, and E2 from Boston Biochem) or expressed and purified from E. coli as described (Mateja et al., 2015 (link)).
8
Protein Extraction and Immunoblotting Protocol
9
Western Blot Analysis of Apoptosis Markers
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Cell lysates were prepared using NP-40 lysis buffer (1% NP-40, 1 mM EDTA, 150 mM NaCl, 50 mM Tris pH 7.4, 1 mM PMSF, Complete Protease Inhibitors [Roche]). Protein content was determined by Bio-Rad assay, proteins were separated by SDS-PAGE and blotted onto nitrocellulose. Membranes were probed with antibodies at 1/1000 dilution unless otherwise stated. BAX (Santa Cruz, sc-493), caspase-3 (Cell Signalling, 9662), PARP1 (eBioscience, 14–6666), actin-HRP (Sigma-Aldrich, A3854), APAF-1 (Merk, AB16941), ZEB-1 (Sigma-Aldrich, HPA027524), MITF (Abcam, ab20663) antibodies followed by incubation with the appropriate HRP conjugated secondary antibody and detection by ECL.
10
Western Blot Analysis of Hypoxia Markers
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Western immunoblotting was performed as reported.21 (link) Primary antibodies used were: RIOK3 (H00008780-M01, Abnova), HIF1α (610959, BD Transduction Laboratories), HIF2α (NB100-122; Novus), CA9 (clone M75, gift of J. Pastorek, Bratislava), TPM3 (ab113692, Abcam), TPM3 (HPA009066, Sigma-Aldrich), TMOD3 (HPA001849, Sigma-Aldrich) and Actin-HRP (A3854; Sigma-Aldrich). Band densitometry was performed using the Analyze Gels tool in ImageJ (http://imagej.nih.gov/ij , version 1.47q).
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