Journal: The Journal of Experimental Medicine

Article Title: Resistance to Granzyme B-mediated Cytochrome c Release in Bak-deficient Cells

doi:

Figure Lengend Snippet: Deficient expression of Bak in a clonal Jurkat cell line. (A) Wild-type or the variant Jurkat cell line, Bak − , were incubated in 1% NP-40 lysis buffer for 30 min at 4°C. The resultant lysates which contained both cytoplasm and mitochondria, were resolved by SDS/PAGE and assessed by immunoblotting for the presence of Bak. Four different anti–human Bak Ab were used for blotting. The membranes were stripped and reprobed for β-actin to demonstrate equal loading. (B) Expression of Bak in mitochondria of wild-type, but not in mitochondria of Bak-deficient Jurkat cells. Expression of Bak was examined in cytosol (S-100), purified mitochondria, or purified mitochondria treated with alkali to remove nonspecifically attached proteins. These cell fractions were resolved by SDS/PAGE and immunoblotted sequentially by Bak-specific Ab-1 and Ab-2. After additional stripping, the membranes were probed with anti–Cox IV Ab, as a marker for mitochondrial fractions, and with anti–β-actin as a marker for cytosolic proteins.

Article Snippet: Anticytochrome c oxidase IV (Cox IV) Ab was from Molecular Probes; rabbit anti-Bid Ab was a gift from Xiaodong Wang (Southwestern Medical Center, Dallas, TX).

Techniques: Expressing, Variant Assay, Incubation, Lysis, SDS Page, Western Blot, Purification, Stripping Membranes, Marker

Journal: The Journal of Experimental Medicine

Article Title: Resistance to Granzyme B-mediated Cytochrome c Release in Bak-deficient Cells

doi:

Figure Lengend Snippet: GrB-mediated cleavage of Bid and its translocation to the mitochondria in extracts of Bak-deficient cells. (A) Jurkat cell lines, including wild-type, Bak-deficient, Neo-, or Bcl-X L –transduced cells were Dounce homogenized, and then treated with GrB (1 μg/ml) for 1 h at 30°C. The extracts were then separated into S-100 cytosol and mitochondria fractions. Loss of full-length Bid was detected in cytosols of all Jurkat cell variants treated with GrB. The anti-BID Ab used for this blot detects only full length Bid. (B) GrB-mediated cleavage of Bid proceeds in the presence of Z-VAD-FMK. Extracts of Bak-deficient Jurkat cells were incubated with Z-VAD-FMK (100 μM) for 20 min before the addition of GrB (1 μg/m) for 1 h at 30°C. The extracts were then fractionated into S-100 and mitochondrial fractions, which were assessed by immunoblotting and sequential probing for the presence of Bid, caspase-3, and β-actin. (C) Translocation of GrB-cleaved Bid to the mitochondria of Bak-deficient Jurkat cells. The mitochondrial fraction of extracts of Bak-deficient cells treated with GrB in the presence or absence of Z-VAD-FMK was assessed by immunoblotting for the presence of tBid. The membrane was stripped and reprobed with anti-Cox IV mAb, as a mitochondrial marker and to demonstrate equal loading.

Article Snippet: Anticytochrome c oxidase IV (Cox IV) Ab was from Molecular Probes; rabbit anti-Bid Ab was a gift from Xiaodong Wang (Southwestern Medical Center, Dallas, TX).

Techniques: Translocation Assay, Incubation, Western Blot, Marker

Journal: Translational Andrology and Urology

Article Title: Suppression of NLRP3 inflammasome activation by astragaloside IV via promotion of mitophagy to ameliorate radiation-induced renal injury in mice

doi: 10.21037/tau-23-323

Figure Lengend Snippet: Animal treatment and the effects of radiation on renal function, ROS level, and expression of Cytc. (A) The timeline shows the time points for drug treatment and radiation. (B) SCr (a), BUN (b), and UA (c) levels in different groups of mice are shown. (C) ROS levels in different groups of mouse kidneys were evaluated by dihydroethidium staining (400×). (D) Western blot expression results and the quantification of the levels of Cytc in cytoplasm (a,c) and mitochondria (b,d) in the kidneys of each group, with normalization to GAPDH and COX-IV respectively. The data are expressed as the mean ± standard error of the mean (n=3; **, P<0.01 and ***, P<0.001 vs. the control group; # , P<0.05, ## , P<0.01 and ### , P<0.001 vs. the DMSO group). ROS, reactive oxygen species; Cytc, cytochrome C; AS-IV, astragaloside IV; CsA, cyclosporin A; SCr, creatinine; BUN, blood urea nitrogen; UA, uric acid; DMSO, dimethyl sulfoxide; IR, irradiation; c-Cyt c, Cytc in the cytosol; COX-IV, Cytc oxidase IV; mito-Cyt c, Cytc in the mitochondria.

Article Snippet: This was followed by blocking in 5% skimmed milk in tris-buffered saline with Tween20 (TBST) at RT for 1 h and overnight incubation with primary antibodies against c-Cytc (bs-0013R; Bioss Antibodies), NLRP3, cleaved caspase-1 (bs-10442R; Bioss Antibodies), IL-1β (bs-0812R; Bioss), Bax (A00183; Boster Bio), B-cell lymphoma-2 (Bcl-2; BA0412; Boster Bio), prostacyclin 62 (P62; bs-2951R; Bioss Antibodies), parkin (bs-23687R; Bioss Antibodies), LC3, PINK1 (23274-1-AP; Proteintech), cleaved caspase-3, cleaved caspase-9 (20750S; Cell Signaling Technology, Danvers, MA, USA), Cytc oxidase IV (COX-IV; 11242-1-AP; Proteintech), β-actin (bs-0061R; Bioss Antibodies), and GAPDH (K200057M, Solarbio) at 4 °C, with subsequent incubation in corresponding HRP-conjugated secondary antibodies at RT for 2 hours.

Techniques: Expressing, Staining, Western Blot, Control, Irradiation