Journal: Scientific Reports

Article Title: One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy

doi: 10.1038/srep14072

Figure Lengend Snippet: TIRF ( a ) and SMLM ( b ) image of mEos2 single-molecule surface. The boxed insets in ( b ) are magnifications of two mEos2 molecules and demonstrate repeated localizations (crosses) of single mEos2 FPs. ( c ) Intensity time traces of mEos2 molecules boxed in ( b ) show distinct blinking after photoactivation before the FPs photobleach irreversibly. Time traces ( c ) and crosses ( b ) having the same color belong to the same molecule. ( d ) Distribution of the number of blinking events N blinks,i upon photoactivation for single mEos2 FPs. The distribution is fit to a geometric distribution with p surface = 0.30 ± 0.01 as fitting parameter (n = 622 mEos2 molecules, adjusted R 2 = 0.995) Note that N blinks,i corresponds to the number of fluorophore reactivations, not total appearances. Scale bars: 1 μm; inset 10 nm.

Article Snippet: For single-molecule mEos2 surfaces, glass slides (PLANO GmbH) were washed in isopropyl alcohol (Sigma) for 30 min, plasma cleaned in N 2 plasma for 10 min (Diener Electronic) and incubated in 100 μg/ml poly-L-lysine solution (Sigma) for 2 h at room temperature.

Techniques:

Journal: Scientific Reports

Article Title: One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy

doi: 10.1038/srep14072

Figure Lengend Snippet: SMLM images of CD86-mEos2 ( a ) and CTLA-4-mEos2 ( b ) at the membrane of Hela cells. ( c , e ) Magnifications of region I or II boxed in ( a ) or ( b ) respectively. The inset shows a magnification of the boxed region. Distributions of N blinks are generated from SMLM data sets. ( d ) For CD86-mEos2, the distribution is well approximated by a geometric distribution and yields p membrane = 0.28 ± 0.01 as blink parameter, which is used as a fixed value for all other membrane proteins (n = 11 cells, adjusted R 2 = 0.986 for CD86-mEos2). ( f ) For CTLA-4-mEos2, the distribution of N blinks fits to a negative binomial distribution with an average number of N = 1.93 ± 0.02 underlying fluorescent probes per complex (n = 11 cells, adjusted R 2 = 0.989 for CTLA-4-mEos2). Scale bars: 2 μm; inset 100 nm.

Article Snippet: For single-molecule mEos2 surfaces, glass slides (PLANO GmbH) were washed in isopropyl alcohol (Sigma) for 30 min, plasma cleaned in N 2 plasma for 10 min (Diener Electronic) and incubated in 100 μg/ml poly-L-lysine solution (Sigma) for 2 h at room temperature.

Techniques: Membrane, Generated

Journal: Scientific Reports

Article Title: One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy

doi: 10.1038/srep14072

Figure Lengend Snippet: VSVG-mEos2 and CD80-mEos2 were expressed in Hela cells and subjected to SMLM imaging. ( a , b ) Resulting distributions of N blinks were fit to negative binomial distributions yielding N = 1.41 ± 0.03 for CD80-mEos2 ( a ) and N = 2.71 ± 0.04 for VSVG-mEos2 ( b ) as fitting parameters (n = 10 cells, adjusted R 2 = 0.983 for CD80-mEos2; n = 11 cells, adjusted R 2 = 0.964 for VSVG-mEos2).

Article Snippet: For single-molecule mEos2 surfaces, glass slides (PLANO GmbH) were washed in isopropyl alcohol (Sigma) for 30 min, plasma cleaned in N 2 plasma for 10 min (Diener Electronic) and incubated in 100 μg/ml poly-L-lysine solution (Sigma) for 2 h at room temperature.

Techniques: Imaging

Journal: Nature Communications

Article Title: GFI1 facilitates efficient DNA repair by regulating PRMT1 dependent methylation of MRE11 and 53BP1

doi: 10.1038/s41467-018-03817-5

Figure Lengend Snippet: GFI1 activities are independent of DNA damage. a GFI1-Flag fusion protein was immunoprecipitated in 293T cells treated with 5 Gy IR and allowed to recover for the indicated amount of time. Extracts were separated by SDS–PAGE and blotted for the indicated proteins. b SupT1 cells were spread on glass slides 15 min and 1 h after irradiation using a Cytospin, stained for endogenous Gfi1 and γ-H2AX and visualized for immunofluorescence by confocal microscopy. Control cells stained without primary antibody but with secondary antibodies are shown. c U2OS cells carrying a LacO array and expressing a LacR-Fok1-mCherry endonuclease were transfected with a vector expressing the GFI1-GFP fusion protein. These cells were plated on cover glass, stained for γ-H2AX and visualized for immunofluorescence by confocal microscopy. d U2OS cells expressing a GFI1-GFP fusion protein were exposed to 405 nm UV micro-irradiation and the recruitment of the GFI1-GFP fusion protein to the site of damage was quantified by confocal microscopy. Average signal intensity is shown with error bars representing s.d. Recruitment of Ku80-mRuby2 fusion protein and GFP protein are shown as controls. Representative images of selected time points are shown on the right. Scale bar represents 10 μm

Article Snippet: Single strand electrophoresis (comet assays) experiments were based on the procedure described by Olive et al . Microscopy slides (Ultident cat. No 170-7107A-S) were coated with 800 μl of 1% low melt agarose (Sigma, A9045) 24 h prior to the assay.

Techniques: Immunoprecipitation, SDS Page, Irradiation, Staining, Immunofluorescence, Confocal Microscopy, Control, Expressing, Transfection, Plasmid Preparation