Dabrafenib, 3-methyladenosine, and 4-phenylbutyrate were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The antibodies to phosphoprotein kinase RNA-like endoplasmic reticulum kinase (PERK), CHOP, inositol-requiring enzyme 1α, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-LC3 I/II was purchased from Novus (St Louis, MO, USA); and anti-p62 was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).
Anti lc3 1 2
Manufactured by Novus Biologicals
Sourced in United States
Anti-LC3 I/II is a primary antibody that recognizes the microtubule-associated protein 1A/1B-light chain 3 (LC3). LC3 is a key autophagy marker that exists in two forms: LC3-I and LC3-II. This antibody detects both forms of LC3 and is commonly used in immunoblotting and immunofluorescence applications to study autophagy processes.
Automatically generated - may contain errors
Lab products found in correlation
Dabrafenib, by Merck Group (1 mentions)
Anti p62, by Santa Cruz Biotechnology (1 mentions)
Inositol requiring enzyme 1α, by Cell Signaling Technology (1 mentions)
4 phenylbutyrate, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Anti cpt1a, by Abcam (1 mentions)
Anti hsp60, by Santa Cruz Biotechnology (1 mentions)
Anti cleaved caspase 9, by Abcam (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti mlkl, by Merck Group (1 mentions)
Anti normal rabbit igg, by Cell Signaling Technology (1 mentions)
Anti mlkl phospho s358, by Abcam (1 mentions)
Anti rab14, by Merck Group (1 mentions)
Anti rab7a, by Merck Group (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Anti atf6, by Cell Signaling Technology (1 mentions)
Anti parp1, by Proteintech (1 mentions)
6 protocols using anti lc3 1 2
1
Endoplasmic Reticulum Stress Pathway
2
Immunoblotting Analysis of Cell Signaling Proteins
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Cells were harvested and disrupted in IP lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 1 mM EDTA, 5% glycerol; Thermo Scientific, MA, USA). Extracted proteins were separated by SDS-PAGE and transferred onto nylon membranes. Binding of primary antibodies was detected using peroxidase-conjugated secondary antibodies. Visualization was performed using the ChemiDoc XRS system with Image Lab software (Bio-Rad, CA, USA). Antibodies included anti-CPT1A, anti-cleaved caspase 9, anti-MLKL (phospho S358) (Abcam, MA, USA), anti-cleaved-PARP, anti-cleaved caspase 3, anti-normal rabbit IgG and anti-normal mouse IgG (Cell Signaling Technologies, MA, USA), anti-Rab14, anti-Rab7A, anti-MLKL and anti-β-actin (Sigma-Aldrich, Darmstadt, Germany), anti-HSP60 (Santa Cruz, CA, USA), and anti-LC3 I/II (Novus Biologicals, CA, USA).
3
Western Blot Analysis of Cellular Markers
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The following primary antibodies were used for Western blot analysis: rabbit polyclonal anti-PARP1(1:1000) (Proteintech, Rosemont, IL, USA, #13371-1), rabbit polyclonal anti-phospho STAT3 Tyr705 (1:500) (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, #sc-8059), mouse monoclonal anti-STAT3 (1:100) (BD Transduction Lab, Franklin Lakes, NJ, USA, #610189), rabbit polyclonal anti-LC3I/II (1:1000) (Novus, CO, USA, #NB100-2220), mouse monoclonal anti-p62/SQSTM1 (1:300) (BD Transduction Lab, #610832), rabbit polyclonal anti-XBP1 (1:1000) (NovusBio, #NBP1-77681SS), rabbit polyclonal anti-ATF6 (1:200) (Cell Signaling Technology, Danvers, MA, USA, #65880), rabbit polyclonal anti-phospho eIF2α (Ser15) (1:200) (Cell Signaling, #3398), rabbit polyclonal anti-eIF2α (1:500) (Cell Signaling, #9722), mouse monoclonal anti-β-actin (1:10000) (Sigma Aldrich, #A5441) and anti hsp70 (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, #sc-32239) were used as loading control. Goat anti-rabbit IgG-horseradish peroxidase HRP (1:10000) (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, sc-2004), goat anti-mouse IgG-horseradish peroxidase HRP (1:10000) (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, sc-2005) were used as secondary antibodies. All primary and secondary antibodies used in this study were diluted in a PBS-0.2% Tween 20 solution containing 3% BSA.
4
Western Blot Analysis of OXPHOS Proteins
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Antibodies anti‐COX4, anti‐UQCRC2, anti‐NDUFA9, anti‐ATP5A, anti‐SDHA were from Abcam (OXPHOS cocktail ab110412, dilution 1:2,000); anti‐SDHB (ab14714, dilution 1:200) and anti‐COX1 (ab14705, dilution 1:2,000) were from Abcam; anti‐HSC70 was from Santa Cruz (sc‐7298, dilution 1:1,000); anti‐GAPDH was from Abcam (ab8245, dilution 1:40,000); anti‐P62 was from Abnova (H00008878‐M01, dilution 1:1,000); anti‐LC3‐I/II was from Novus Biologicals (NB100‐2220, dilution 1:1,000); anti‐LAMP1 was from Cell Signaling (3243, dilution 1:1,000); anti‐PINK1 was from Novus (BC100‐494, dilution 1:1,000); and anti‐Parkin was from Abcam (ab77924, dilution 1:500).
5
Investigating Stem Cell Protein Regulation
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pMSCs treated under different conditions were collected and then lysed with RIPA buffer along with PMSF protease inhibitor. The primary antibodies used for immunodetection included anti-OCT4 (Cat.#: AF0226; Affinity Biosciences, Changzhou, China), anti-p53 (Cat.#: AF0879; Affinity Biosciences, Changzhou, China; Cat.#: 10442-1-AP; Proteintech, Wuhan, China), anti-p62 (Cat.#: ab109012; Abcam, Cambridge, MA, USA), anti-LC3-I/II (Cat.#: NB100-2220; Novusbio, CO, USA), anti-p-AMPK (Cat.#: AF3423; Affinity Biosciences, Changzhou, China), anti-AMPK (Cat.#: sc74461; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (Cat.#: sc8432; Signalway Antibody, Baltimore, MD, USA). The specific protein bands were visualized with the Odyssey Infrared Imaging System (LI-COR, Lincoln, Dearborn, MI, USA). The band density was analyzed using Image-Pro Plus 5.1 software (MEDIA CYBERNETICS, Silver Spring, MD, USA) using β-actin as an internal control and then normalized to the vehicle control.
6
Subcellular Fractionation and Western Blot
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Different cells were harvested and fractionated to produce cytosolic and nuclear lysates using the NE-PER kit (Thermo Fisher Scientific). For whole cell lysates, cells were lysed in RIPA buffer. Both protease and phosphatase inhibitors were included in the respective lysis buffer. ~47 μg of protein was separated on a 4%-15% gradient SDS-polyacrylamide gel (Bio-Rad) which were transferred to nitrocellulose membranes, stained with Ponceau S stain, washed, blocked with 5% non-fat milk, and incubated overnight at 4°C with the following primary antibodies: anti-α-Tubulin (Cell Signaling Technology (C.S.T.) #2144), anti-Lamin A/C (C.S.T. #4777), anti-SON (Abcam #121033 and LSBio LS-C803664), anti-YAP1 (C.S.T. #12395), anti-GFP (C.S.T. #2956), anti-Tau (Sigma-Aldrich, #A0024), anti-p-Tau (a gift from Dr. Peters Davies), anti-β-actin (C.S.T. #4970), anti-puromycin (BioLegend 381502), anti-ubiquitin (C.S.T. #58395), anti-LC3-I/II (C.S.T. #2775), anti-p62 (C.S.T. #23214), anti-ATF4 (C.S.T. #11815), anti-ATF6 (Novus 70B1413.1), and anti-XBP1s (BioLegend 658802). Membranes were treated with the appropriate secondary antibody conjugated to horseradish peroxidase the following day and then ECL Prime Western Blotting Detection Reagent (Cytiva) was applied. A Bio-Rad ChemiDoc MP Imaging System was used to visualize the signal, and signal intensities were determined with ImageJ 112 (link).
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