All chemicals were obtained from Sigma-Aldrich, except OptiPrep (Axis-Shield), DCF (Thermo Fisher Scientific), and Percoll (GE Healthcare). Antibodies were purchased from Sigma-Aldrich against TMX1 and LC3 (rabbit antihuman); from EMD Millipore against SERCA2b (mouse antihuman) and against total as well as phosphorylated AMPK (rabbit antihuman); from Thermo Fisher Scientific against PDI, CHOP, and actin (mouse antihuman); from BD against BiP/GRP78 and p62 (mouse antihuman); from Abcam against FACL4 (goat antihuman), mitochondrial complex II (mouse antihuman), and PGC-1α (rabbit antihuman); and from Cell Signaling Technology. The antibodies against anti-myc (Invitrogen) and the FLAG tag (Rockland) were purchased as indicated. We thank L. Berthiaume (University of Alberta, Alberta, Canada) for the goat anti–human cytochrome c. The affinity-purified rabbit anti-TMX1 antiserum was generated by 21st Century Biochemicals using a peptide corresponding to amino acids 251–271 (AESKEGTNKDFPQNAIRQRSL). The rabbit anti–calnexin antibody has been previously described (Myhill et al., 2008 (link)). HeLa cells were from ECACC, and A375P cells were from E. Sviderskaya (St. George’s, University of London, London, England, UK). Melanoma tissue samples were purchased from the Alberta Tumor Bank.
Bip grp78
Manufactured by Abcam
Sourced in United States, United Kingdom
BiP/GRP78 is a molecular chaperone protein that is a central regulator of endoplasmic reticulum (ER) function. It plays a key role in protein folding and quality control in the ER.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (3 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (3 mentions)
Las4000 imager, by GE Healthcare (3 mentions)
Tissue protein extraction reagent, by Thermo Fisher Scientific (3 mentions)
Sirt7 and foxo1 acetyl lys294, by LifeSpan BioSciences (3 mentions)
Ncor2 smrt, by Abcam (3 mentions)
Sirt5, by LifeSpan BioSciences (3 mentions)
Las 4000, by Cytiva (3 mentions)
Percoll, by GE Healthcare (1 mentions)
Anti myc, by Thermo Fisher Scientific (1 mentions)
Optiprep, by Axis-Shield (1 mentions)
Pgc 1α, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pcr purification kit, by Promega (1 mentions)
Caspase 4, by Abcam (1 mentions)
Gel extraction kit, by Promega (1 mentions)
Calreticulin, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Ym155, by Selleck Chemicals (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
8 protocols using bip grp78
1
Comprehensive Antibody Analysis for Cellular Signaling
2
Survivin-Modulated Cell Apoptosis Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PCR Purification Kit, Plasmid Mini Kit, and Gel Extraction Kit were from Promega (Madison, WI, USA). HiFiFast DNA polymerase was obtained from BiovisuaLab (Shanghai, China). Lipofectamine 2000 was from Invitrogen (Carlsbad, CA, USA). Survivin expression plasmid was constructed by the Sangon Biotech (Shanghai, China). YM155 was purchased from Selleck chemicals (Houston, TX, USA). Antibodies to IL-24, calreticulin, survivin, phosphorylated JNK and c-Jun, caspase-4, BiP/GRP78, and β-actin were obtained from Abcam (Cambridge, UK). All chemicals and reagents were purchased from Sigma unless noted specifically.
3
Mitochondrial Protein Fractionation and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The left ventricular tissue of hearts and H9c2 cells were harvested after diverse treatments and prepared for western blotting. The separation of cytosol and mitochondrial protein fraction from cardiac tissues and H9c2 cells were obtained by using a mitochondria isolation kit (Beyotime, Shanghai, China) according to the manufacture's instruction. After separating the protein samples by SDS‐PAGE, the proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% nonfat milk in TBST. The membrane was then incubated with primary antibodies at 4°C overnight. The antibodies against Bip/GRP78 (1: 1,000), IRE1α (1: 1,000), Total OXPHOS, irisin, and p‐ IRE1α (1: 1,000) were purchased from Abcam while the antibodies against XBP‐1s (1:1000), ATF4 (1:1000), CHOP (1:1000), Caspase 3 (1:1000), Bax (1:1000), Bcl‐2 (1:1000), Cyto C (1:1000), and GAPDH (1:5000) were purchased from Cell Signaling Technology. After wash with TBST, the membranes were incubated with HRP‐conjugated second antibodies for 1.5‐2 h at RT. Then the proteins were visualized using chemiluminescent reagents (Millipore, Billerica, MA, USA) under ChemiDoc Imaging System (Bio‐Rad Laboratories, Hercules, CA, USA) and the densities of the bands were quantified by Image Lab software (Bio‐Rad Laboratories, Hercules, CA, USA). GAPDH was used as an internal reference.
4
Quantitative Analysis of UPR Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative real-time PCR was performed as described above. The ribosomal protein gene Rpl19 served as an endogenous control for quantitation of UPR-related genes. For RT-PCR, total RNA was extracted from transfected cells using a Total RNA kit (Omega, USA). Each reaction used 20 ng of total RNA as a template, and the primers used were selected according to a previous study52 (link). Western blot analysis was also performed according to a previous study52 (link), and a rabbit polyclonal antibody against BiP/Grp78 (Abcam, UK) was used as the primary antibody.
5
Western Blot and Co-immunoprecipitation Analysis of Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues and cells were homogenized in Tissue Protein Extraction Reagent or Mammalian Protein Extraction Reagent (#78510 or 78505, Thermo Fisher Scientific, Waltham, MA, USA). Nuclear and cytoplasmic extracts were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (#78835, Thermo Fisher Scientific). Homogenates (20 μg for Western blot and 200 μg for co-IP) were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% skim milk, blots were probed with primary antibodies (Supplementary Table 3 ) against PAK4, p-PAK4 (S474), HSP90, ubiquitin, HMGCS2, phospho-PKA Substrate, phospho-(Ser/Thr), Sirt1, Sirt6, phospho-Akt (Ser473), Akt, acetylated-Lysine, CHOP, phospho-S6 Ribosomal Protein (Ser240/244), S6 Ribosomal Protein, NCoR1, NEDD4, ATF-6, phospho-PERK (Thr980) (Cell Signaling Technology), PAK4, FGF21, and PPARα (Santa Cruz Biotechnology), Sirt4, lamin B1, and GAPDH (Bioworld Technology, St Louis Park, MN, USA), Sirt5, Sirt7 and FoxO1 (Acetyl-Lys294) (LifeSpan Biosciences, Seattle, WA, USA), and Sirt2, Sirt3, GRP78 BiP, NCOR2/SMRT, CPT1α or CPT1A,T-OXPHOS, THRβ, p-IRE1α and MDM2 (Abcam, Cambridge, UK), LXRα (Proteintech, Rosemont, IL, USA). Immunoreactive bands were detected with an LAS-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA).
6
Western Blot and Co-immunoprecipitation Analysis of Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues and cells were homogenized in Tissue Protein Extraction Reagent or Mammalian Protein Extraction Reagent (#78510 or 78505, Thermo Fisher Scientific, Waltham, MA, USA). Nuclear and cytoplasmic extracts were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (#78835, Thermo Fisher Scientific). Homogenates (20 μg for Western blot and 200 μg for co-IP) were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% skim milk, blots were probed with primary antibodies (Supplementary Table 3 ) against PAK4, p-PAK4 (S474), HSP90, ubiquitin, HMGCS2, phospho-PKA Substrate, phospho-(Ser/Thr), Sirt1, Sirt6, phospho-Akt (Ser473), Akt, acetylated-Lysine, CHOP, phospho-S6 Ribosomal Protein (Ser240/244), S6 Ribosomal Protein, NCoR1, NEDD4, ATF-6, phospho-PERK (Thr980) (Cell Signaling Technology), PAK4, FGF21, and PPARα (Santa Cruz Biotechnology), Sirt4, lamin B1, and GAPDH (Bioworld Technology, St Louis Park, MN, USA), Sirt5, Sirt7 and FoxO1 (Acetyl-Lys294) (LifeSpan Biosciences, Seattle, WA, USA), and Sirt2, Sirt3, GRP78 BiP, NCOR2/SMRT, CPT1α or CPT1A,T-OXPHOS, THRβ, p-IRE1α and MDM2 (Abcam, Cambridge, UK), LXRα (Proteintech, Rosemont, IL, USA). Immunoreactive bands were detected with an LAS-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA).
7
Western Blot and Co-immunoprecipitation Analysis of Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues and cells were homogenized in Tissue Protein Extraction Reagent or Mammalian Protein Extraction Reagent (#78510 or 78505, Thermo Fisher Scientific, Waltham, MA, USA). Nuclear and cytoplasmic extracts were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (#78835, Thermo Fisher Scientific). Homogenates (20 μg for Western blot and 200 μg for co-IP) were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% skim milk, blots were probed with primary antibodies (Supplementary Table 3 ) against PAK4, p-PAK4 (S474), HSP90, ubiquitin, HMGCS2, phospho-PKA Substrate, phospho-(Ser/Thr), Sirt1, Sirt6, phospho-Akt (Ser473), Akt, acetylated-Lysine, CHOP, phospho-S6 Ribosomal Protein (Ser240/244), S6 Ribosomal Protein, NCoR1, NEDD4, ATF-6, phospho-PERK (Thr980) (Cell Signaling Technology), PAK4, FGF21, and PPARα (Santa Cruz Biotechnology), Sirt4, lamin B1, and GAPDH (Bioworld Technology, St Louis Park, MN, USA), Sirt5, Sirt7 and FoxO1 (Acetyl-Lys294) (LifeSpan Biosciences, Seattle, WA, USA), and Sirt2, Sirt3, GRP78 BiP, NCOR2/SMRT, CPT1α or CPT1A,T-OXPHOS, THRβ, p-IRE1α and MDM2 (Abcam, Cambridge, UK), LXRα (Proteintech, Rosemont, IL, USA). Immunoreactive bands were detected with an LAS-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA).
8
Immunocytochemical Characterization of Astrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fixed LP and HP AbAs were permeabilized with 0.1 % Triton X-100 during 30 min, washed 3 times with PBS and blocked 30 min with 5 % bovine serum albumin (BSA). Then cells were incubated with one or two of the following antibodies: 1:500 anti-GLT1 (Cell Signaling, #3838); 1:600 anti-S100β (Sigma, S2532); 1:500 anti-GS (abcam, 49873); 1:300 anti-MMP-2 (Thermo Fisher Scientific, 436000), 1:500 anti-MMP-9 (Thermo Fisher Scientific, PA5-13199) or 1:300 anti-78-kDa glucose-regulated protein (GRP78/BiP, abcam, ab-21685). After a 4 °C overnight incubation and 3 washes with PBS, cells were incubated for 90 min with 1:800 dilutions of 1 mg/mL secondary antibodies conjugated to Alexa fluorescent probes (Thermo Fisher Scientific). Then cells were washed and mounted in 50 % glycerol containing 1 μg/mL 4′, 6-diamino-2-phenylindole (DAPI). As negative controls, the primary or secondary antibodies were omitted. A part of the cells were labeled with 1:500 Phalloidin-FITC (Thermo Fisher Scientific, F432), rinsed 2 times with PBS and mounted in glycerol/DAPI. All of the cells were imaged between 24 h and 48 h after the labeling procedure was completed.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!