Sample preparation and immunoblotting conditions were performed as previously described [19 (link)]. Cell and striatum protein extracts from one hemisphere of mice were prepared in cell lysis buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton-X100 and 0.1% SDS) supplemented with phosphatase and protease inhibitors (Thermo Scientific; Waltham, MA, USA). Protein samples were separated on 4–20% SDS Criterion TGX Stain-Free gels (BioRad; Hercules, CA, USA) and transferred to a nitrocellulose membrane (BioRad 0.2 µm). Primary antibodies used are as follows: anti-GAPDH (Santa Cruz, sc-365062, 1:10,000; Dallas, TX, USA), anti-HSF1 (Bethyl, A303-176A, 1:1000; Waltham, MA, USA), anti-PSD-95 (Novus, NB300-556, 1:1000; Centennial, CO, USA), and anti-Hsp70 (Enzo, C92F3A-5, 1:1000; Farmingdal, NY, USA). Quantitative analyses were performed using ImageJ software and normalized to GAPDH controls.
C92f3a 5
Manufactured by Enzo Life Sciences
Sourced in United States
The C92F3A-5 is a laboratory equipment designed for performing various scientific experiments and analysis. It is a multi-functional device capable of carrying out specific tasks. The core function of this product is to facilitate precise and controlled experimentation in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Nb300 556, by Novus Biologicals (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Hsp70, by Enzo Life Sciences (1 mentions)
β actin, by Merck Group (1 mentions)
Spa 901, by Enzo Life Sciences (1 mentions)
Sc 9996, by Santa Cruz Biotechnology (1 mentions)
Ab32157, by Abcam (1 mentions)
A5441, by Merck Group (1 mentions)
L7543, by Merck Group (1 mentions)
Sc 200, by Santa Cruz Biotechnology (1 mentions)
Dsirna, by Integrated DNA Technologies (1 mentions)
Ab8226, by Abcam (1 mentions)
Mms 101r, by Fortrea (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Doxycycline dox, by Merck Group (1 mentions)
Ver 155008, by Merck Group (1 mentions)
Ab109018, by Abcam (1 mentions)
Ab53039, by Abcam (1 mentions)
7 protocols using c92f3a 5
1
Immunoblotting Techniques for Protein Analysis
2
Isolation and Quantification of HSP70 in PBMCs
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Following the previously described centrifugation of Histopaque and whole blood, the buffy coat containing the PBMCs was pipetted into a conical tube containing 10-mL of phosphate-buffered saline (Sigma-Aldrich 4417, St. Louis, MO). The mixture was centrifuged (968 g, 10 min, 20°C). The supernatant was disposed of and the pellet was stored at -80°C until analysis. HSP70 concentration in PBMCs was measured via ELISA with a detection range of 0.78–50 ng/mL (C92F3A-5, Enzo Life Sciences, Inc., East Farmingdale, New York). The pellet was resuspended in 1x extraction reagent, centrifuged (×21,000g, 10 min, 4°C), and the supernatant (cell lysate) was transferred to sterile microtubes, following the manufacturer's instructions. A 4-fold dilution was used and the samples were loaded onto a precoated 96-well microplate. All reagents were added and washed according to the manufacturer's instructions.
3
Protein Extraction and Western Blot Analysis
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Mouse intestinal tissues were physically minced in Pro-prep solution (17081, Intron Inc., Korea), and then proteins were extracted from the lysates. Total proteins were extracted from cells using RIPA lysis buffer. Western blot analysis was performed using the following antibodies: HSP70 (1:1000, C92F3A-5, Enzo Life Sciences, NY), HSF1 (1:1000, sc-17757, Santa Cruz Biotechnology), eNOS (1:1000, sc-634, Santa Cruz Biotechnology), cleaved caspase-3 (1:1000, #9961, Cell Signaling, MA), cleaved PARP (1:1000, #9542 Cell Signaling), and β-actin (1:3000, Sigma, MO). The blots were incubated with these antibodies overnight at 4°C.
4
Immunoblotting Analysis of HSF1 Oligomerization
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Cellular proteins were extracted with lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxicholate, 0.1% SDS, 1 mM dithiothreitol (DTT), 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF)) supplemented with a protease inhibitor cocktail (Complete). In some experiments, whole cell extracts (43 (link),44 (link)) were used to assess levels of HSF1. Protein concentration in cell lysates was determined by a Bradford-based protein assay. A total of 25 μg of proteins were resolved by SDS-PAGE, transferred to a PVDF membrane and analyzed by immunoblotting using mouse anti-human HSP72 monoclonal antibody C92F3A-5, rabbit anti-human HSF1 polyclonal antibody SPA-901 or rat anti-mouse HSF2 monoclonal antibody SPA-960 (all from Enzo). A mouse anti-human GAPDH monoclonal antibody 9484 (Abcam) was used as a loading control. HSF1 oligomerization was assessed using amine-specific cross-linker ethylene glycol bis-succinimidyl succinate (EGS) (Pierce). Whole cell extract (50 μg) prepared as previously described (43 (link),44 (link)) was incubated with 0.5 mM EGS for 30 min at RT. The cross-linking reaction was quenched by the addition of 50 mM glycine/0.025 mM Tris, pH 7.5 and incubation for 15 min at RT. Proteins were fractionated through a 6% SDS-PAGE gel and analyzed by immunoblotting using anti-HSF1 antibody SPA-901.
5
Antibody Panel for UPR Markers
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Commercially available primary antibodies against the following antigens were used: eIF2α phosphorylated at Ser51: rabbit monoclonal, 119A11 (Cell Signaling) and rabbit monoclonal, ab32157 (Abcam); total eIF2α: rabbit monoclonal, D7D3 (Cell Signaling); LC3B: rabbit polyclonal, L7543 (Sigma); HSP70: mouse monoclonal, C92F3A-5 (Enzo Life Sciences); GFP: mouse monoclonal (sc-9996, Santa Cruz); ATF4: rabbit polyclonal, sc-200 (Santa Cruz); CHOP: mouse monoclonal, clone L63F7 (Cell Signaling); TIAR: mouse monoclonal, 610352 (BD Biosciences); cleaved caspase 3: rabbit polyclonal, 9661S (Cell Signaling); puromycin: mouse monoclonal, clone 12D10 (Merck Millipore); beta-actin: mouse monoclonal, A5441 (Sigma). Antibodies were used at 1:1000 dilution for all applications.
6
Antibodies and Reagents for CFTR Research
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SiRNA was purchased from Qiagen, dSiRNA were obtained from Integrated DNA Technologies, and the target sequences are listed in S1 Table
7
Molecular Interactions and Regulation of CHIP and Hsp70
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CHIP or CHIP mutants or Hsp70 or Hsp70 mutants were cloned into pcDNA3.1 or p3x Flag-CMV-10 (Sigma) or pET28a (Novagen). The Myc-MDM2 expressing plasmid was a kind gift from Dr. A.G. Jochemsen. Myc-Itch was obtained from Dr. Tony Pawson. Flag-Itch was obtained from Dr. A. Atfi. His-Ub is a kind gift from Dr. W. Gu. All PCR products were confirmed by sequencing. Doxycycline (Dox, D9891, Sigma), Cycloheximide (CHX, 01810, Sigma), Ver-155008 (SML0271, Sigma), and Hepatocyte growth factor (HGF, H9661, Sigma). Anti-p63 [p63 (ΔNp63 (N-16), sc-8609R, Santa Cruz Biotechnology; ab53039, Abcam; Poly 619002 (anti-p63 (ΔN), Poly 618902 (anti-TAp63), 938102 (anti-TAp63), BioLegend], anti-CHIP [H-231 and C10, Santa Cruz Biotechnology; EPR4447 (ab134064), Abcam], anti-Hsp70 (C92F3A5, Enzo Life Sciences), anti-Itch (ab109018, Abcam), anti-Mdm2 (2A10, Calbiochem; SMP14, BD Biosciences), anti-PRP19 (ab27692, Abcam), anti-p21 (F-5, Santa Cruz), anti-Myc (9E10, Roche), anti-Flag (M5, M2, Sigma), anti-HA (12CA5, Roche), anti-ubiquitin (550944, BD Biosciences), anti-actin (A2066, Sigma), anti-T7 tag (69522–4, Novagen) and anti-p53 (Pab1801, Santa Cruz Biotechnology) were used according to the manufacturer's instructions.
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