Journal: Nature communications

Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

doi: 10.1038/ncomms2236

Figure Lengend Snippet: Localization of ErbB2 in mitochondria. (A) Cytosolic, nuclear, mitochondrial, and plasma membrane proteins were isolated and subjected to SDS-PAGE followed by probing with indicated antibodies. Two exogenous ErbB2 overexpressing breast cancer cell lines MCF7/ErbB2 and MDA-MB-231 (left), and two natural ErbB2-positive breast cancer cell lines SKBR3 and BT474 (right) were used for Western Blotting. VDAC1 and prohibitin were mitochondrial markers; Integrin β1 and IGF1Rα were plasma membrane markers; α-Tubulin and ERK were cytoplasmic markers; KDEL was an ER marker; EEA1 was an early endosomes marker; Golgi complex was a marker for the detection of Golgi; LAMP2 was an lysosome marker and Lamin B1 was a nucleus marker. (B) MCF7 cells, mouse heart and liver tissues, and ErbB2 positive and negative breast cancer patient samples were analyzed by Western blotting. (C) Co-localization of ErbB2 and mitochondria. Mitochondria were stained with Mitotracker-Green in ErbB2 transfected MCF7 cells. The cells were fixed and incubated with antibodies against ErbB2 (mouse), followed by incubation of monoclonal mouse Anti–Cy3 antibody (red). Images were analyzed with Nikon NIS-Elements AR software. Green: mitochondria; Red: ErbB2; Yellow: Co-localization of ErbB2 and mitochondria. The lower panel contains images with a higher magnification. Scale bars: 20 μm. (D) Localization of ErbB2 inside mitochondria. Intact mitochondria of SKBR3 cells were isolated and treated with Protease K at 0.5 mg/ml for 15 min at room temperature. 1% Triton was added into the reaction and 2 mM PMSF was added to stop the reaction. Each reaction was started with an equal amount/volume of mitochondria lysate. Mitochondrial lysate then was loaded onto 8% SDS PAGE followed by blotting with anti-ErbB2, anti-BCL-2 and anti-Prohibitin antibodies.

Article Snippet: Protease K was purchased from Sigma; the immunoprecipitation assay was conducted using Pierce Co-immunoprecipitation Kit (Cat: 26149) from Thermo Scientific.

Techniques: Isolation, SDS Page, Multiple Displacement Amplification, Western Blot, Marker, Staining, Transfection, Incubation, Software

Journal: Redox Biology

Article Title: Redox-dependent condensation of the mycobacterial nucleoid by WhiB4

doi: 10.1016/j.redox.2018.08.006

Figure Lengend Snippet: Mtb WhiB4 exhibits non-specific DNA binding activities. (A) 150 ng of either linear (L) or supercoiled (SC) plasmid DNA was incubated with oxidized apo-WhiB4 (250 ng [lanes 2, 6]; 300 ng [lanes 3, 7]; 350 ng [4, 8]). Lanes 1 and 5 represent DNA without WhiB4. (B) Binding of apo-WhiB4 (100, 150, 200, 250, 300 and 350 ng) with 1 kb λ-DNA ladder (λ) (150 ng). (C) WhiB4 represses transcription . In vitro transcribed pGEM plasmid in the absence of apo-WhiB4 (lane 1), oxidized apo-WhiB4 in complex with pGEM (lane 2), in vitro transcription of pGEM in the presence of oxidized (lane 3) or reduced (lane 4) apo-WhiB4. The reactions in lane 3 and 4 were treated with 6% SDS and 4 mg ml -1 protease K to remove WhiB4 from the mixture before separation. C: pGEM plasmid DNA alone. Cysteine residues regulate DNA binding of WhiB4 (D) DTT-treatment abolished DNA binding of WhiB4. Oxidized apo-WhiB4 (350 ng) was treated with DTT (0, 200, 400, 800 mM) for 2 h followed by binding to the supercoiled plasmid DNA. C: represents DNA without WhiB4. (E) Purified wt apo-WhiB4 and Cys mutant variants were treated with thiol oxidant, diamide, and the gel-shift assay was performed as described earlier. DNA binding assays using Mtb HU (50–350 ng) and Mtb TcrX (150–350 ng) with (F) supercoiled DNA and (G) 1 kb λ-DNA ladder. (H) 350 ng of Mtb HU or (I) Mtb TcrX were treated with increasing concentrations of DTT (0, 200, 400, 800 mM) for 2 h followed by binding to the supercoiled plasmid DNA. The samples were analyzed on 1% agarose gel.

Article Snippet: Samples were treated with 6% SDS (Amresco Inc.) and 4 mg ml-1 proteinase K (Amresco Inc.) for 30 min at 37o C and the reaction products were analyzed on a 1% agarose gel.

Techniques: Binding Assay, Plasmid Preparation, Incubation, In Vitro, Purification, Mutagenesis, Electrophoretic Mobility Shift Assay, Agarose Gel Electrophoresis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prion-inducing domain 2-114 of yeast Sup35 protein transforms in vitro into amyloid-like filaments

doi:

Figure Lengend Snippet: Protease K resistance of Sup35pN filamentous aggregates ( a ) and of amorphous aggregates ( b ) as determined by SDS/PAGE. The protease K concentration in each reaction is indicated.

Article Snippet: Two microliters of protease K (Boehringer Mannheim no. 1413 783) solution in TE buffer at concentrations from 0.5 to 4.0 μg/ml were added and the mixture was incubated at 37°C for 80 min.

Techniques: SDS Page, Concentration Assay