Journal: Chemistry and physics of lipids

Article Title: Single-particle cryo-EM studies of transmembrane proteins in SMA copolymer nanodiscs

doi: 10.1016/j.chemphyslip.2019.03.007

Figure Lengend Snippet: Cryo-EM of the alternative complex III: cyt c oxidase supercomplex in SMA nanodiscs. A, 3D reconstruction of the supercomplex. The ACIII is colored in blue while the cyt c oxidase is colored in yellow. Also shown is a smoothed transparent surface of the SMA nanodisc at a lower density value. This panel has been reproduced with permission from Springer Nature. B, 3D reconstruction of the supercomplex colored by local resolution in angstrom.

Article Snippet: Only the portion of the cyt c oxidase component of the supercomplex that is near the interface of ACIII is well resolved, but a complete model of supercomplex can be assembled from the cryoEM data by using the existing X-ray structure of related cyt c oxidases.

Techniques: Cryo-EM Sample Prep

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