Figure 2. Slimb localizes to centrioles.... | Rockefeller University Press

Figure 2.

$Figure 2. Slimb localizes to centrioles. (A) Immunoblot of affinity-purified anti-Slimb antibody against an S2 cell lysate. (B) slimb RNAi depletes protein by 98%. (C and C′) Immunostaining of Slimb (green, monochrome) and D-PLP centrioles (red) in interphase S2 cells. (D and D′) Stable S2 line expressing Slimb-EGFP (green, monochrome) and mCherry–SAS-6 centrioles in live interphase cells. (C–D′) Centrioles (arrowheads) are shown at a higher magnification (insets). (E) Endogenous Slimb and Slimb-EGFP cosediment with centrioles purified from S2 cells on a 20–70% sucrose gradient. Fractions were immunoblotted for D-PLP, GFP, and Slimb. Asterisks denote peak centriole-containing fractions. (F) Slimb (green, arrowheads) immunolocalizes to D-PLP centrioles (red) after 24-h drug-induced S-, G2-, or M-phase arrest. Histograms (to scale) of DNA content assessed by HTM (5,000 cells/histogram). Condensed DNA (blue) reveals a mitotic cell. Insets show centrioles at a higher magnification. (G) Graph of endogenous Slimb levels after 24-h drug-induced cell cycle arrest as determined from quantitative anti-Slimb immunoblots (below graph). (B and G) α-Tubulin was used as a loading control. Bars, 5 µm.$

Slimb localizes to centrioles. (A) Immunoblot of affinity-purified anti-Slimb antibody against an S2 cell lysate. (B) slimb RNAi depletes protein by 98%. (C and C′) Immunostaining of Slimb (green, monochrome) and D-PLP centrioles (red) in interphase S2 cells. (D and D′) Stable S2 line expressing Slimb-EGFP (green, monochrome) and mCherry–SAS-6 centrioles in live interphase cells. (C–D′) Centrioles (arrowheads) are shown at a higher magnification (insets). (E) Endogenous Slimb and Slimb-EGFP cosediment with centrioles purified from S2 cells on a 20–70% sucrose gradient. Fractions were immunoblotted for D-PLP, GFP, and Slimb. Asterisks denote peak centriole-containing fractions. (F) Slimb (green, arrowheads) immunolocalizes to D-PLP centrioles (red) after 24-h drug-induced S-, G2-, or M-phase arrest. Histograms (to scale) of DNA content assessed by HTM (5,000 cells/histogram). Condensed DNA (blue) reveals a mitotic cell. Insets show centrioles at a higher magnification. (G) Graph of endogenous Slimb levels after 24-h drug-induced cell cycle arrest as determined from quantitative anti-Slimb immunoblots (below graph). (B and G) α-Tubulin was used as a loading control. Bars, 5 µm.