Immunoblotting experiments were conducted as previously described (15 (link)). Antibodies used for immunoblotting were specific for the following proteins: LC3b, PERK, IRE1α, P-AMPK, AMPK (total), P-ULK (Ser757), P-ULK (Ser317), ULK (total), COXIV, cytochrome c, calnexin, HSP60, mitofusin-1, and Bcl-2 (Cell Signaling Technology); BAP31, ATF6, Tom40, Tom22, prohibitin, and VDAC1 (Santa Cruz Biotechnology); Parkin and FACL-4 (Abcam); and NDUFS4, NDUFB11, α-tubulin, β-actin, and flag (Sigma-Aldrich). Antibodies were diluted to 1:1000, except for NDUFS4 (1:500) or anti–β-actin (1:10,000). Secondary antibodies were purchased from Promega (anti-rabbit and anti-mouse at 1:5000).
Prohibitin
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
Prohibitin is a protein that functions as a molecular chaperone, assisting in the proper folding and assembly of other cellular proteins. It plays a role in regulating cellular processes such as cell proliferation and mitochondrial function. Prohibitin is widely expressed in various tissues and cell types.
Automatically generated - may contain errors
Lab products found in correlation
Ab109381, by Abcam (2 mentions)
Ab40660, by Abcam (2 mentions)
Ab33770, by Abcam (2 mentions)
Ab86695, by Abcam (2 mentions)
Ab52640, by Abcam (2 mentions)
Ab68166, by Abcam (2 mentions)
Rac1 activity assay kit, by Cell Biolabs (2 mentions)
P erk, by Cell Signaling Technology (1 mentions)
α tubulin, by Merck Group (1 mentions)
Mitofusin 1, by Cell Signaling Technology (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
Tom22, by Santa Cruz Biotechnology (1 mentions)
Cox 4, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Cytochrome c, by Cell Signaling Technology (1 mentions)
Vdac1, by Santa Cruz Biotechnology (1 mentions)
Calnexin, by Cell Signaling Technology (1 mentions)
P ulk, by Cell Signaling Technology (1 mentions)
Hsp60, by Cell Signaling Technology (1 mentions)
7 protocols using prohibitin
1
Immunoblotting Experimental Protocol
2
Comprehensive Molecular Techniques Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bcl2, APE1, PCNA, prohibitin and tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). MTT cell growth kit was obtained from Sigma (St. Louis, MO). QIAamp DNA isolation kit was purchased from Qiagen (Chatsworth, CA). 4,6-diamidino-2-phenylindole (DAPI), QD605 goat anti-rabbit IgG conjugate (red), QD705 goat anti-mouse IgG conjugate (green) and Pico Green dsDNA Quantitation kit were obtained from Invitrogen (Carlsbad, CA). HEX-5ʹ-end-labeled 26-mer duplex oligonucleotide (5ʹ-AAT TCA CCG GTA CCF CCT AGA ATT CG-3’) was purchased from IDT Technologies (Coralville, IA). LA PCR Kit and TaKaRa ExTak PCR kit were obtained from Clontech Laboratories, Inc (Mountain View, CA). All of the reagents used were obtained from commercial sources unless otherwise stated.
3
Molecular Mechanisms of GBE-Mediated Cytoprotection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GBE was provided by Wanbangde Pharmaceutical Group Co., Ltd. Chemicals such as L-glutamate, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DCFH-DA and DMSO were obtained from Sigma-Aldrich; Merck KGaA. Antibody were obtained from Santa Cruz Biotechnology, Inc. or Abcam: p-SRC (ab40660, Abcam), SRC (ab109381, Abcam), p-VAV2 (ab86695, Abcam), Vav2 (ab52640, Abcam), p-p66Shc (ab68166, Abcam), p66Shc (ab33770, Abcam), cytochrome c (ab133504, Abcam), β-actin (sc-58673, Santa Cruz Biotechnology, Inc.), prohibitin (sc-377037, Santa Cruz Biotechnology, Inc.), Goat Anti-Rabbit IgG H&L (HRP) (ab7090, Abcam), Goat Anti-Mouse IgG H&L (HRP) (ab205719, Abcam). Rac1 activity assay kit was obtained from Cell Biolabs. Amplex Red hydrogen peroxide/peroxidase assay kit was obtained from Thermo Fisher Scientific, Inc. Caspase-3 Activity assay kit was obtained from Abcam. Other chemicals and reagents used in this study were obtained from Beyotime.
4
Reconstitution and analysis of AMPK complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For in vitro reconstitution, each sample was prepared with 30 ul 2x laemmli sample buffer. Purified lysosomes were checked with LAMP2 (abcam ab25631), EEA1 (CST 3288), Prohibitin (Santa Cruz 28259), PDI (Santa Cruz 20132), and Histone H3 (Huaxingbio, HX1850) antibodies. Myc tagged proteins were detected with anti-Myc antibody (ImmunoWag YM3203). LAMP1-RFP-FLAG labeled lysosomes were probed with Flag antibody (Sigma F7425). T172 site phosphorylated AMPKα subunit and total AMPKα subunit were detected with p-AMPKα (CST 2535) and AMPKα (CST 2532) antibodies respectively. STRAD (Santa Cruz sc-34102) and MO25 (CST 2716 s) antibodies were used to detect Myc-LKB1-STRAD-MO25 complex.
For detection of AAK-2 or RSKS-1 phosphorylation, worms at indicated stage were lysed with 2x laemmli sample buffer equal to the volume of worm pellet. AAK-2 phosphorylation was probed with human p-AMPKα (T172) antibody (CST 2535). RSKS-1 phosphorylation was probed with human p-S6K1 (T389) antibody (CST 9205). Tubulin (abcam ab6161) was used as loading control.
For detection of AAK-2 or RSKS-1 phosphorylation, worms at indicated stage were lysed with 2x laemmli sample buffer equal to the volume of worm pellet. AAK-2 phosphorylation was probed with human p-AMPKα (T172) antibody (CST 2535). RSKS-1 phosphorylation was probed with human p-S6K1 (T389) antibody (CST 9205). Tubulin (abcam ab6161) was used as loading control.
5
Investigating Oxidative Stress Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chemicals such as RAC1 inhibitor NSC23766 and NOX (NADPH oxidase) inhibitor VAS2870 were purchased from MCE Biotechnology. MTT (3-(4,5-Dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) cell proliferation assay kit was purchased from Solarbio. Reagents such as antibodies were purchased from Abcam, Santa Cruz Biotechnology and NOVUS: p-SRC (ab40660, Abcam), SRC (ab109381, Abcam), p-Vav2 (ab86695, Abcam), Vav2 (ab52640, Abcam), p-p66SHC (ab68166, Abcam), p66SHC (ab33770, Abcam), GAPDH (ab8245, Abcam), Prohibitin (sc-377037, Santa Cruz Biotechnology), NOX4 (NB110-58849, NOVUS), GFAP (ab7260, Abcam). RAC1 activity assay kit was purchased from Cell Biolabs. NOX activity assay kit, MDA (malondialdehyde) assay kit and SOD (super oxide dismutase) activity assay kit were purchased from Solarbio and Abcam. Other chemicals and reagents used in this study were obtained from Beyotime and Sangon.
6
Mitochondrial Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondria was isolated from homogenized eyes using Mitochondria Isolation Kit for Tissue (Thermo Fisher Scientific, Waltham, USA). Isolated mitochondria was loaded on 12% gel prepared using TGX ™ FastCast ™ Acrylamide Kit (Bio-Rad, Hercules, USA), and isolated proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Bio-Rad) using Trans-Blot Turbo Transfer system (25 V and 1A for 30 min: Turbo-Standard). The membrane was blocked with 2.5% Blotting-Grade Blocker (Non-fat dry milk, Bio-Rad) in TBST buffer (20 mM Tris, 150 mM NaCl, and 0.1% Tween ® 20; pH 7.5) for 1 h at 26 °C followed by overnight incubation at 4 °C with two primary antibodies: anti-OxPhos (1:500 dilution; Anti-OxPhos complex kit; Invitrogen, Carlsbad, USA) and prohibitin (1:500 dilution; Santacruz, Dallas, USA). Further, the membrane was washed several times with TBST buffer and incubated with goat anti-mouse IgG (H + L) secondary antibody (1:3000 dilution) with horseradish peroxidase-conjugate (Invitrogen) for 1.5 h at 26 °C. The protein complexes were visualized using Clarity ™ Western ECL Substrate (Bio-rad) on ChemiDoc System from Bio-rad. The samples were analyzed using Image Lab 6.0. (Bio-rad).
7
Western Blot Analysis of Key Cellular Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of total and nuclear extracts was performed as described elsewhere [28] . Western blot analysis was performed using antibodies against total and phospho-Akt (Ser 473 ), adenylate cyclase, BACE1, CHOP, total and phospho-CREB (Ser 133 ), total and phospho-eIF2, GRP78/BiP, insulin receptor -subunit (IRNRF1, total and phospho-PKA (Thr 197 ), total and p-STAT3 (Tyr 705 )(Cell Signaling Technology Inc., Danvers, MA), OXPHOS (Mito Sciences, Eugene, OR), A42 (Biolegend), sAPP (Covance, Alnwick, UK), PGC-1Abcam, Cambridge, United Kingdom), GAPDH, IBlamin B, Oct-1, p65, PPAR, PPAR/, prohibitin (Santa Cruz), total and phospho-IRS-1 (Ser 307 ) (Millipore, Billerica, MA) and -actin (Sigma, St. Louis, MO). Detection was achieved using the Western Lightning® Plus-ECL chemiluminescence kit (PerkinElmer, Waltham, MA). The equal loading of proteins was assessed by Ponceau S staining. The size of detected proteins was estimated using protein molecular-mass standards (Bio-Rad, Hercules, CA). For validation, we used a protein marker (Precision Plus Protein Dual Color Standards 1610374; Bio-Rad, Hercules, CA, USA), on the same blots. All of these commercially available antibodies showed a single distinct band at the molecular weight indicated in the datasheets.
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!