NP cells were plated on glass coverslips and treated with 100 nM MitoTracker™ Red CMXRos (Thermo Fisher, M7512) for 30 min after completion of the experimental treatments. Cells were then fixed with 4% PFA or ice cold methanol for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. and blocked with 1% BSA for 1 h. Cells were incubated with anti-BNIP3 (3769S), anti-BNIP3L (12396S), anti-LC3 (12741) (Cell Signaling), anti-TOMM20 (ab186734), anti-LAMP1 (ab24170), anti-PINK1 (ab23707) (Abcam), anti-PARKIN (sc-32282) (Santa Cruz), anti-DRP1 (611113), anti-OPA1 (612607) (BD Biosciences), anti-BECLIN1 (NB500–249) (Novus Biologicals) in blocking buffer at 1:100–200 at 4°C overnight. After washing, cells were incubated with Alexa Flour-488 or Alexa Flour 647 and mounted with ProLong Gold Antifade Mountant with DAPI. Negative controls were used to confirm the specificity of staining (Suppl. Fig. 4 ). Cells were visualized using a Nikon A1R confocal microscope using 60x objective (CFI plan Apo Lambda 60x/1.40 oil). Positive staining for markers and co-localization of LC3 and LAMP1 with mitochondria was measured as area (pixel2/cell) using the colocalization Plugin of ImageJ software (http://rsb.info.nih.gov/ij/ ).
Anti parkin sc 32282
Manufactured by Santa Cruz Biotechnology
Sourced in France
Anti-Parkin (sc-32282) is a primary antibody produced in mouse that recognizes the Parkin protein. Parkin is an E3 ubiquitin-protein ligase involved in the ubiquitination and degradation of proteins. This antibody can be used for the detection of Parkin in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti pink1 ab23707, by Abcam (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Anti drp1, by BD (1 mentions)
Anti rip1, by BD (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Zvad fmk, by Bachem (1 mentions)
Anti tom20 sc 11415, by Santa Cruz Biotechnology (1 mentions)
Lsm 710 meta, by Zeiss (1 mentions)
Anti lc3, by Merck Group (1 mentions)
Anti cyclophilin b 11607 1 ap, by Proteintech (1 mentions)
Anti tom20 sc 17764, by Santa Cruz Biotechnology (1 mentions)
Anti lc3b l7543, by Merck Group (1 mentions)
Carbonyl cyanide m chlorophenylhydrazone cccp, by Merck Group (1 mentions)
Gapdh ab128915, by Abcam (1 mentions)
5 protocols using anti parkin sc 32282
1
Mitochondrial Dynamics and Autophagy Imaging
2
Induction of Regulated Cell Death Pathways
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Recombinant human TNF, produced and purified to at least 99% homogeneity in our laboratory, has a specific biological activity of 3 × 107 IU/mg. Agonistic anti-human Fas (clone 2R2) was from Cell Diagnostica (Munster, Germany). zVAD-fmk was from Bachem (Bubendorf, Switzerland). Nec-1 was from Calbiochem (San Diego, CA, USA). The Tak1 inhibitor NP-009245 was purchased from AnalytiCon Discovery GmbH (Potsdam, Germany). Ac-DEVD-amc was from PeptaNova GmbH (Sandhausen, Germany). CCCP, Rotenone and Antimycin-A were purchased from Sigma Aldrich (Steinheim, Germany). NSA was from Toronto Research Chemicals (Toronto, ON, Canada). The Drp1 inhibitor mdivi was from Merck Chemicals Ltd (Nottingham, UK). Anti-β-actin (clone C4; MP BiomedicalsEurope NV, Illkirch, France), anti-Parkin (sc-32282) and anti-MLKL (sc-130172) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-RIP1 from BD Biosciences (610459; Franklin Lakes, NJ, USA), and anti-RIP3 (R4277) and anti-MLKL (M6697 and SAB1408428) from Sigma Aldrich. Anti-Drp-1 was from BD Transduction laboratories (clone 8/DLP1, Franklin Lakes, NJ, USA).
3
Immunocytochemical Analysis of Mitochondria and Autophagy Markers
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GCs were grown on coverslips in 12-well plates for 4 days and then exposed to the treatments described above. After washing in PBS, the cell climbing sheets were fixed with 4% paraformaldehyde (PFA) for 1 h, permeabilized using 0.5% Triton X-100 in PBS for 10 min at 4 °C, and blocked with 1% BSA for 1 h at room temperature. The coverslips were then incubated with 1:100 dilutions of anti-Tom20 (sc-11415; Santa Cruz), anti-Parkin (sc-32282; Santa Cruz), or anti-LC3 (L7543; Sigma) for 1 h at 37 °C. They were subsequently stained for another 1 h with rabbit or mouse Alexa Fluor 488 (A-11008), 568 (A-11031), and 633 (A-21072) secondary antibodies at 1:200 dilutions (Invitrogen). The coverslips were washed, mounted on slides, and observed under a laser-scanning confocal microscope (Zeiss LSM 710 META).
4
Mitochondrial Protein Profiling Protocols
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The antibodies used in this study were as follows: anti‐OGG1 (NB100‐106) and anti‐PINK1 (8E10.1D6) from Novus (Novus Biologicals), anti‐Parkin (sc‐32282) and anti‐TOM20 (sc‐17764) from Santa Cruz, anti‐LC3 (12741), and anti‐BCL2 (3498 S) from CST, anti‐COXIV (ab202554) from Abcam, anti‐α‐tubulin (11224‐1‐AP), anti‐BAX (60267‐1‐Ig), and anti‐cyclophilin B (11607‐1‐AP) from ProteinTech. All the secondary antibodies were purchased from Thermo Fisher Scientific. The specific inhibitor, Th5487, of OGG1 (HY‐125276) was purchased from Med Chem Express.
5
Mitochondrial Protein Analysis Reagents
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Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and propidium iodide (PI) were purchased from Sigma. The following antibodies were used for western blot analysis or immunofluorescence: COX IV (4850), NDP52 (60732), and GST (2625) were purchased from Cell Signaling Technology; monoclonal anti-ubiquitin (sc-8017), anti-Tom20 (sc-17764), anti-PINK1 (sc-517353), and anti-Parkin (sc-32282) were purchased from Santa Cruz; anti-LC3B (L7543) was purchased from Sigma; and anti-Tim23 (611222) was purchased from Becton Dickinson and Company. Actin (EM21002) was purchased from HuaBio. GAPDH (ab128915) was purchased from Abcam.
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