Western blot analysis was conducted as described previously(23 (link)). Antibodies used were phospho-S473-KAP1 (644602, BioLegend), phospho-S824-KAP1 (A300-767A, Bethyl), KAP1 (A300-274A, Bethyl), β-actin (MAB1501R), H3K27me3 (07-449) from Millipore, Tom20 (sc-17764) and GAPDH (sc-25778) from Santa Cruz, phospho-T172-AMPKα (2535), AMPKα (2532), phospho-T180/Y182-p38 (9216) and p38 (9212) from Cell Signaling, MFN1 (GTX116254), MFN2 (GTX102055), OPA1 (GTX48589) and FIS1/TTC11 (GTX111010) from GeneTex, H3 (39763), H3K9me3 (61013) from Active Motif and Mitoprofile OXPHOS Cocktail (MS601) from MitoSciences.
Tom20 sc 17764
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Tom20 (sc-17764) is a primary antibody that targets the translocase of outer membrane (TOM) 20 protein. TOM20 is a subunit of the TOM complex, which is responsible for the import of proteins into the mitochondrial outer membrane.
Automatically generated - may contain errors
Lab products found in correlation
Phospho s824 kap1 a300 767 a, by Fortis Life Sciences (1 mentions)
Phospho s473 kap1, by BioLegend (1 mentions)
Gapdh sc 25778, by Santa Cruz Biotechnology (1 mentions)
Synaptopodin, by Progen Biotechnik (1 mentions)
Ant063, by Antgene (1 mentions)
Calreticulin ab92516, by Abcam (1 mentions)
Ant030, by Antgene (1 mentions)
P erk, by Santa Cruz Biotechnology (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Ab54481, by Abcam (1 mentions)
Ab48394, by Abcam (1 mentions)
β actin 4970, by Cell Signaling Technology (1 mentions)
Model lsm880 confocal microscope, by Zeiss (1 mentions)
Lc3 14600 1 ap, by Proteintech (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
5 protocols using tom20 sc 17764
1
Comprehensive Western Blot Analysis
2
Quantitative Colocalization Analysis of Mitochondrial and Endoplasmic Reticulum Proteins
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The immunofluorescence assay analyses using the frozen renal biopsies from patients, the frozen kidney sections from rats and the cell climbing films were performed as previously described. The following primary antibodies were used in this study: Mfn2 (1:200; Abcam, United Kingdom); WT1 (NB110-60011, 1:50; Novus, GER); Synaptopodin (65194, 1:50; Progen, GER); TOM20 (sc-17764; 1:50; Santa Cruz, United States); Calreticulin (ab92516; 1:100; Abcam, United Kingdom); PERK (1:50; Santa Cruz, United States). The Alex Fluor 488/594 donkey anti-rabbit/Mouse IgG (HL) (ANT023, ANT024, ANT029, ANT030; 1:100; Antgene, Wuhan, China) were used as secondary antibodies. The nuclei were stained with an anti-fluorescence quencher containing DAPI (ANT063; Antgene, Wuhan, China). A confocal microscope (Olympus, Japan) was used to observe all of the microscopic images. The Pearson’s correlation coefficients (PCC) have been calculated in immunofluorescence assay of TOM20 and CRT, which with a value between 1 and −1, was used to quantitative colocalization between two proteins (1 means perfect correlation; −1 means completely excluded; 0 means no relationship) (Schober et al., 2018 (link)).
3
Molecular Mechanisms of Cadmium-Induced Stress
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Cadmium chloride (202908), Melatonin (M5250), Chloroquine diphosphate (C6628) were from Sigma Chemical Co (St. Louis, MO). Antibodies against HSP60 (12165S), COX IV (4850S), LC3B-I/II (3868S), ATF4 (11815S), p-eIF2α (9721S), eIF2α (2103S), GCN2 (3302S) and β-Actin (4970S) were from Cell Signaling Technology (Beverley, MA). Antibodies against CYP11A1 (SC18043) and TOM20 (SC17764) were from Santa Cruz (California, US). Antibodies against BNIP3 (ab10433), PGC1α (ab54481) and LC3B-I/II (ab48394) were from Abcam (Cambridge, MA). The secondary antibody conjugated with Alexa Fluor 488 (711-545-152) and the secondary antibody conjugated with Cy3TM (715-165-150) were from Jackson ImmunoResearch (PA, USA). Progesterone (P4) ELISA kit (E-EL-0090C) was from Elabscience (Wuhan, China). Human short interfering RNAs targeting BNIP3 and GCN2 were from GenePharma (Shanghai, China).
4
Immunofluorescence Microscopy for Subcellular Organelles
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Cells were grown on coverslips before treatment. Cells were washed in PBS, fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% TritonX-100 in PBS for 3 min, and blocked in 2% BSA in PBS for 30 min. Primary antibodies -TOM20 (sc-17764, Santa Cruz), PHB2 (ab182139, abcam), LC3 (14600–1-AP, Proteintech, Manchester, UK)- were used at 1:200 overnight at 4 °C, followed by secondary antibodies at 1:1000 and DAPI (0.5 µg/ml) for 1 h at room temperature. Anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 647 were obtained from Molecular Probes (Invitrogen, Carlsbad, CA, USA). Cells were mounted with Fluoromount G (SouthernBiotech, Birmingham, AL, USA). Images were acquired with a Carl Zeiss model LSM880 confocal microscope and a Apochromat 63×/1.4 Oil M27 objective lens and they were collected using 405, 488, and 561 nm laser lines for excitation and appropriate emission filters. Images were analyzed with Image J Software. Pearson’s coefficient was obtained with JACoP plugin [26 (link)].
5
Antibody Sources for Western Blot
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T7 tag (69-048-3MI) antibody was purchased from Novagen. GST (sc-138), LDH (sc-33781), FLOT1 (sc-74566), NIX (sc-166314), TUFM (sc-393924), and TOM20 (sc-17764) antibodies were from Santa Cruz Biotechnology. Flag (M2, F3165) and HA (3F10, 11867423001) antibodies were from Sigma-Aldrich. GABARAPL1 (66458–1), β-Actin (60008–1), Lamin B1 (12987–1), and MT-ND1 (19703–1) antibodies were from Proteintech. V5 tag (R960-25) antibody was from Thermo Fisher Scientific. LC3B (NB100-2220) antibody was from Novus Biologicals. LC3A (4599), LC3C (14736), GABARAPL1 (26632), GABARAPL2 (14256S), VDAC (4661), Flag tag (14793), and V5 tag (13202) antibodies were from Cell Signaling Technology. P62/SQSTM1 (PM045) and GABARAP (PM037) antibodies were from MBL International. MT-CO2 antibody (ab79393) was from Abcam. LAMP1 antibody (11215-R107) was from Sino Biological. VDAC (600-101-HB2) antibody was from Rockland. Anti-RTA and rabbit anti-vIRF-1 were a gift from Gary Hayward, and rat anti-vIRF-1 (residues 1–22) was custom-generated from Thermo Fisher.
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