The total protein of mouse breast tissues and MAC-T cells was separated by RIPA reagent (Biosharp, China). The BCA Protein Assay Kit (Thermo Scientific, MA, USA) was performed to detect protein concentration. The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); then, polyvinylidene difluoride (PVDF) membranes were used to receive the transferred protein from the gel. The membranes were blocked with 5% skimmed milk for 2 h; then, the primary antibodies (BiP, Ero1-Lα, PDI, IRE1α, PERK, CHOP, JNK, ERK, p38, p65, and β-actin from Cell Signaling Technology, USA; PPARγ from Abcam, UK) were incubated at 4°C overnight, and then, the secondary antibody (anti-rabbit IgG and HRP-linked antibody from Cell Signaling Technology, USA) was incubated at 37°C for 1 h. Finally, the immunoblot signal was displayed with ECL ultrasensitive chemiluminescent solution with chemiluminescence imaging system.
Ero1 lα
Manufactured by Cell Signaling Technology
Sourced in United States
Ero1-Lα is a recombinant protein that functions as an endoplasmic reticulum (ER) oxidoreductin-1-like alpha enzyme. It plays a role in the oxidative protein folding process within the ER.
Automatically generated - may contain errors
Lab products found in correlation
Calnexin, by Cell Signaling Technology (7 mentions)
Ire1α, by Cell Signaling Technology (6 mentions)
P erk, by Cell Signaling Technology (6 mentions)
Eif2α, by Cell Signaling Technology (5 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Grp94, by Cell Signaling Technology (3 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Cleaved parp, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Phospho eif2α, by Cell Signaling Technology (2 mentions)
β actin ac 15, by Merck Group (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Trypan blue, by Avantor (2 mentions)
Nitrocellulose membrane, by Avantor (2 mentions)
Sheep anti mouse igg hrp linked polyclonal antibody, by Avantor (2 mentions)
Donkey anti rabbit igg hrp linked polyclonal antibody, by Avantor (2 mentions)
Ezblock protease inhibitor cocktail, by Avantor (2 mentions)
Atf 6 65 880 s perk 5683 s phospho perk thr980 3179 s ire 1α 3294 s bip 3183 s chop 5554 s pdi, by Cell Signaling Technology (2 mentions)
17 protocols using ero1 lα
1
Protein Expression Analysis in Mouse Tissues
2
CYT997 Protocol for Cellular Assays
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CYT997 (MF: C24H30N6O2, MW: 434.53, purity: 99.46%) was purchased from Selleckchem (Houston, TX, USA) and dissolved in dimethyl sulfoxide (DMSO) to prepare a 40 mM stock solution, which was stored at − 80 °C. DMSO was obtained from Sigma-Aldrich (St. Louis, MO, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), N-acetylcysteine (NAC), 3-methyladenine (3-MA), chloroquine (CQ) and GSK2606414 were purchased from Sigma-Aldrich. EN460 was purchased from MedchemExpress (Monmouth Junction, NJ, USA). Antibodies against PARP, C-PARP, CASPASE-4, LC3B-I/II, BECLIN-1, PERK, P-PERK, EIF2A, P-EIF2A, CHOP, ERO1-Lα, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA).
3
Western Blot Analysis of Cellular Stress Markers
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Total cellular protein lysates were prepared with Pierce lysis buffer (Pierce, Rockford, IL, USA) and quantified by the Bradford method. Equal amounts of total protein (20 µg) were loaded onto 10% SDS‐PAGE gels and electrophoresed at 100 V for 1 hour. Then, the separated proteins were transferred onto PVDF membranes (Millipore, Billerica, MA, USA) at 80 V for 2 hours. The membranes were blocked in 5% non‐fat milk in 1× TBST and then incubated with primary antibodies against EGFR (1:500; Santa Cruz, USA), PERK, IRE1α, ATF 6, (1:1000; Abcam), phospho‐eIF2α, GRP78, GRP94, PDI, ERO1‐Lα, CHOP, phospho‐ATM, DNA‐PK, LC3B, Atg3, cleaved caspase 3, cleaved PARP, and β‐actin (1:1000; Cell Signaling Technology, Boston, MA, USA) at 4°C overnight. After washing with 1× TBST, the membranes were incubated with secondary anti‐mouse or anti‐rabbit IgG HRP‐linked antibodies (1:5000; Cell Signaling Technology) at room temperature for 2 hours. The blots were developed with Target LumiGLO (Cell Signaling Technology) and photographed with DNR BioImaging System (DNR, Israel).
4
Western Blot Analysis of Protein Signaling
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After drug treatment, total protein lysates were prepared, separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane for Western blotting as described previously50 (link). The following antibodies were used: Cell Signaling Technology (MA, USA): BAX (#5023), Bcl-xL (#2764), PERK (#5683), phospho-PERK (Thr980, #3179), Calnexin (#2679), eIF2-α (#5324), phospho-eIF2-α (Ser51, #3398), PDI (#3501), ero1L-α (#3264), BiP (#3177), IRE1α (#3294), MEK1/2 (#9126), phospho-MEK1/2 (Ser217/221, #9154), MEK2 (#9125) ERK1/2 (#4695), phospho-ERK1/2 (Thr202/Tyr204, #4370). Sigma-Aldrich: β-actin (AC-15; #A1978). Primary antibodies were used 1:1000. Secondary, anti-mouse (#7076) and anti-rabbit (#7074) horseradish peroxidase-labeled antibodies from Cell Signaling Technologies were used in working dilutions of 1:10,000.
5
Endoplasmic Reticulum Stress Response
6
Protein Expression Analysis by Western Blotting
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The changes in protein expression induced by the combination were evaluated using Western blotting. The cells were treated under the indicated conditions for 48 h, and whole-cell lysates were obtained using radioimmunoprecipitation assay buffer. Equal amounts of proteins were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After the membranes were blocked by 5% skimmed milk, they were incubated overnight with anti-acetylated histone (Abcam, Cambridge, UK), anti-cyclin D1, anti-glucose-regulated protein 78 (GRP78), anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-cleaved poly(ADP-ribose) polymerase (PARP), anti-endoplasmic oxidoreductin-1-like protein alpha (Ero1-Lα) (Cell Signaling Technology, Danvers, MA, USA), or anti-actin (Millipore, Billerica, MA, USA) primary antibodies. They were then incubated with horseradish-tagged secondary antibodies (Bio-Rad, Hercules, CA, USA). The bands were visualized by chemiluminescence with the ECL Plus system (GE Healthcare, Wauwatosa, WI, USA) according to the manufacturer’s instruction.
7
Oxidative Stress Signaling Pathway
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Small interfering RNAs (siRNAs) for NOX4, NOX1, Smad4, and ASK1 and antibodies against glyceraldehyde 3-phosphate dehydrogenase, phospho-HSP27 (Ser78), HSP27, Trx, GSTM1, AP1, SP1, Smad4, ASK1, and phospho-ASK1 (Thr845) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p38, phospho-p38 (Thr180/Tyr182), phospho-ERK (Thr202/Tyr204), phospho-src (Tyr416), phospho-Akt (Ser473), phospho-stat3 (Tyr705), phospho-p65 (Ser536), phospho-JNK (Thr183/Tyr185), phospho-Smad2 (Ser465/467), phospho-Smad3 (Ser423/425), Smad2, Smad3, p65, ERK, Akt, stat3, JNK, src, BIP, Calnexin, Ero1-Lα, CHOP, PERK, and PDI were purchased from Cell Signaling Technology (Beverly, MA, USA). Phospho-IRE1α was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Peroxiredoxin (Prx) was purchased from Abfrontier (Seoul, Korea). Glutaredoxin (Grx) was purchased from Novus Biologicals (Littleton, CO, USA). Recombinant human TGF-β1 and TGF-β2 were purchased from R&D Systems (Minneapolis, MN, USA). SB203580 was purchased from EMD Millipore (Billerica, MA, USA), and GKT137831 was purchased from ApexBio (Houston, TX, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
8
Investigating ER Stress Signaling Pathways
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We used antibodies against acetylated α-tubulin (Cell Signaling Technology, Danver, MA, USA, #5335), detyrosinated α-tubulin (Sigma, MAB5566), Ero1-Lα (Cell Signaling Technology, #3264), calnexin (Cell Signaling Technology, #2679), BiP (Cell Signaling Technology, #3177), IRE1α (Cell Signaling Technology, #3294), phospho-IRE1α (Cell Signaling Technology, #3398), PERK (Cell Signaling Technology, #5683), phospho-PERK (Abcam, Cambridge, MA, USA, #ab192591), ATF6 (Abcam, #ab227830), and GAPDH (Santa Cruz Biotech, TX, USA, #sc32233). Tunicamycin (Sigma, T7765) and Y-27632 (Sigma, Y0503) were purchased from Sigma-Aldrich. Blebbistatin was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada).
9
Immunoblotting Analysis of ER Stress Markers
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Primary antibodies against PDI (rabbit monoclonal; 1:1000), Ero1-Lα (rabbit monoclonal; 1:1000), IRE1α (rabbit monoclonal; 1:1000), Calnexin (rabbit monoclonal; 1:600), eIF2α (rabbit polyclonal; 1:800), P-eIF2α (rabbit monoclonal; 1:600), and CHOP (mouse monoclonal; 1:800) were purchased from Cell Signaling Technology, Frankfurt am Main, Germany. Antibodies NADPH 4 oxidase (rabbit polyclonal; 1:800), and Total OXPHOS cocktail antibody (rodent monoclonal; 1:800) were purchased from Abcam, Cambridge, UK. Hsp70 (mouse monoclonal; 1:800), and HO-1 (mouse monoclonal; 1:800) were purchased from Enzo life sciences, Lörrach, Germany. β-actin (rabbit polyclonal; 1:1200) from Novusbio, Centennial, CO, USA, and Vinculin (mouse monoclonal, 1:500) from AbD Serotec, Puchheim, Germany. Where applicable secondary antibodies used during immunoblotting were Donkey anti rabbit (1:8000; Novex), Horse anti mouse (1:2000; Cell Signaling Technology), Donkey anti mouse (1:5000; Bethyl), Goat anti rabbit (1:2000; Cell Signaling Technology), and Donkey anti rabbit (1:4000; Novex).
10
Endoplasmic Reticulum Stress Response
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17-AAG (AAJ66960-EX3), AUY-922 (101756–820),17-DMAG (102513–662), RIPA buffer (AAJ63306-AP), Trypan Blue (76180–676), sheep anti-mouse IgG HRP-linked polyclonal antibody (95017–554), donkey anti-rabbit IgG HRP-linked polyclonal antibody (95017–556), EZBlock™ protease inhibitor cocktail (75837–938) and nitrocellulose membranes (10063–173) were obtained from VWR (Radnor, PA). The ATF-6 (65,880 s), PERK (5683 s), Phospho-PERK (Thr980) (3179 s), IRE-1α (3294 s), BiP (3183 s), CHOP (5554 s), PDI (2446 s), and Ero1-Lα (3264 s) antibodies were purchased from Cell Signaling (Danvers, MA). The β-actin (A5441–0.5 ml) antibody was purchased from Sigma-Aldrich (St Louis, MO). The phospho-IRE1α antibody (Ser724) (16927) was obtained from Thermo Fisher Scientific (Waltham, MA).
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