Figure S5. TLNRD1 interacts with CCM2.... | Rockefeller University Press

Figure S5.

$TLNRD1 interacts with CCM2. (A) U2OS cells expressing mito-PDCD10-mScarlet, mito-CCM2-mScarlet, or mito-TLNRD1-mScarlet were imaged using a spinning disk confocal microscope. (B) U2OS cells treated with siRNA targeting KRIT1 or siRNA control. KRIT1 levels were then analyzed using Western blots. A representative western blot is displayed. (C) U2OS cells treated with siRNA targeting KRIT1 or siRNA control and expressing TLNRD1-GFP and mito-CCM2-mScarlet were imaged using a spinning disk confocal microscope. 3D colocalization analyses were performed using the JACoP Fiji plugin, and results are displayed as Tukey boxplots (three biological repeats, n > 21 image stacks per condition). (D) Glutathione agarose-bound GST-CCM2 (beads: B) was incubated with recombinant TLNRD1, TLNRD14H, or TLN1R7R8 (input: I). After multiple washes, proteins bound to the beads (pellet: P) were visualized. The various fractions were then analyzed using SDS-PAGE followed by Coomassie staining. A representative gel of three independent repeats is displayed. Red boxes highlight areas of interest in the gel. (E) U2OS cells expressing various GFP-tagged CCM2 constructs and mito-TLNRD1-mScarlet or mito-mScarlet (CTRL) were imaged using a spinning disk confocal microscope. Representative single Z-planes are displayed. The yellow squares highlight magnified ROIs. Scale bars: (main) 25 µm and (inset) 5 µm. (F) 3D colocalization analyses were performed using the JACoP Fiji plugin, and results are displayed as Tukey boxplots (three biological repeats, n > 38 image stacks per condition). The whiskers (shown here as vertical lines) extend to data points no further from the box than 1.5× the interquartile range. For all panels, the P values were determined using a randomization test. NS indicates no statistical difference between the mean values of the highlighted condition and the control. Source data are available for this figure: SourceData FS5.$

TLNRD1 interacts with CCM2. (A) U2OS cells expressing mito-PDCD10-mScarlet, mito-CCM2-mScarlet, or mito-TLNRD1-mScarlet were imaged using a spinning disk confocal microscope. (B) U2OS cells treated with siRNA targeting KRIT1 or siRNA control. KRIT1 levels were then analyzed using Western blots. A representative western blot is displayed. (C) U2OS cells treated with siRNA targeting KRIT1 or siRNA control and expressing TLNRD1-GFP and mito-CCM2-mScarlet were imaged using a spinning disk confocal microscope. 3D colocalization analyses were performed using the JACoP Fiji plugin, and results are displayed as Tukey boxplots (three biological repeats, n > 21 image stacks per condition). (D) Glutathione agarose-bound GST-CCM2 (beads: B) was incubated with recombinant TLNRD1, TLNRD1^4H, or TLN1^R7R8 (input: I). After multiple washes, proteins bound to the beads (pellet: P) were visualized. The various fractions were then analyzed using SDS-PAGE followed by Coomassie staining. A representative gel of three independent repeats is displayed. Red boxes highlight areas of interest in the gel. (E) U2OS cells expressing various GFP-tagged CCM2 constructs and mito-TLNRD1-mScarlet or mito-mScarlet (CTRL) were imaged using a spinning disk confocal microscope. Representative single Z-planes are displayed. The yellow squares highlight magnified ROIs. Scale bars: (main) 25 µm and (inset) 5 µm. (F) 3D colocalization analyses were performed using the JACoP Fiji plugin, and results are displayed as Tukey boxplots (three biological repeats, n > 38 image stacks per condition). The whiskers (shown here as vertical lines) extend to data points no further from the box than 1.5× the interquartile range. For all panels, the P values were determined using a randomization test. NS indicates no statistical difference between the mean values of the highlighted condition and the control. Source data are available for this figure: SourceData FS5.