Mouse anti cytochrome c

Cytochrome c, Maneb (Manganese ethylene-1,2-bisdithiocarbamate), paraquat (1,1-dimethyl-4,4-bipyridinium), paraformaldehyde, recombinant α-synuclein, Triton X-100, and TRI Reagent were from Sigma (St. Louis, MO). 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) was obtained from Dojindo Laboratories (Rockville, MD) and used without further purification. Chicken polyclonal and mouse monoclonal anti-DMPO antibodies were developed in our laboratory and used in the immuno-spin trapping studies. Rabbit polyclonal anti-tyrosine hydroxylase antibody was from Millipore (Billerica, MA). Rabbit anti-α-synuclein, mouse anti-cytochrome c, anti-complex IV, anti-caspase-3, anti-caspase-9 and anti-β-actin antibodies were from Abcam (Cambridge, MA). Micro BCA Protein Assay Kit, Permount, RIPA buffer, Surfact-Amps X-100, Mitochondria Isolation Kits for Tissue, and Pierce Classic Immunoprecipitation Kits were from Thermo Scientific (Rockford, IL). Nitrocellulose membranes, Prolong Gold anti-fade reagent with DAPI and AlexaFluor secondary antibodies were from Invitrogen (Grand Island, NY). The Vectastain Elite ABC kit for immunohistochemistry was from Vector Laboratories (Peterborough, UK). RNeasy mini kit columns were from Qiagen (Valencia, CA). Agilent Whole Mouse 4X44K microarray slides and the reagents used in microarray experiments were all from Agilent Technologies (Santa Clara, CA).

Parasite proteins isolated from different subcellular fractionations were separated on SDS-polyacrylamide gels and were transferred to polyvinylidene difluoride (PVDF) membranes as previously described (48 (link)). The primary antibodies used were rabbit anti-PfRad51 (1:5,000 dilution) (25 (link)), rabbit anti-PfBlm (1:5,000) (27 (link)), rabbit anti-PfalMre11 (1:5,000) (26 (link)), rabbit anti-HsHistone 3 (1:5,000; Imperial Life Sciences), mouse anti-cytochrome c (1:5,000; Abcam), rabbit anti-GFP (1:5,000; Abcam), and mouse anti-GAPDH (1:5,000; Abcam). The membranes were washed with Tris-buffered saline with Tween 20 (TBS-T) and treated with horseradish peroxidase (HRP)-conjugated anti-mouse (1:10,000; Santa Cruz Biotechnology Inc.) and anti-rabbit (1:10,000; Promega) secondary antibody for 2 h at 4°C. After washes with TBS-T, membranes were developed with a chemiluminescent horseradish peroxidase (HRP) substrate (SuperSignal West Pico Plus; Thermo Scientific) and imaged by using a ChemiDoc Touch imaging system from Bio-Rad.

Protocols have been extensively described recently (Lucocq et al., 2001 (link), Karanasios and Ktistakis, 2015 (link)). In brief, cells on coverslips were fixed in 3.7% Formaldehyde, permeabilized in 0.1% NP40 and stained in a blocking solution containing fish gelation and 0.05% NP40. Antibodies used and their final dilution are as follows: rabbit anti-OPTN (Cayman Chemicals), 1:100; mouse anti-WIPI2 (Bio-Rad), 1:200; rabbit anti-phospho(S172)TBK1 (Cell Signalling), 1:50; mouse anti-Ubiquitin FK2 (Enzo), 1:100; rabbit anti-FIP200 (ProteinTech), 1:100; rabbit anti-ATG9 (Cell Signalling), 1:100; mouse anti-TOMM20 (Abcam), 1:100; rabbit anti-ATG13 (Sigma), 1:100; rabbit anti-LC3 (Sigma), 1:150; rabbit anti-NDP52 (GeneTex), 1:100; rabbit anti-Tax1BP1 (ProteinTech), 1:100; rabbit anti-VAPA (Sigma), 1:200; rabbit anti-VAPB (Sigma), 1:200; mouse anti-EEA1 (BD Biosciences), 1:70; mouse anti-LAMP2 (Developmental Studies Hybridoma Data Bank), 1:200; mouse anti-TUBULIN (Sigma), 1:300; mouse anti-cytochrome C (Abcam), 1:200; rabbit anti-phospho(S351)p62 (kind gift from Dr Masaaki Komatsu), 1:100; rabbit anti-TRAF2 (Abcam) 1:100.