Figure 1. Coupling RNase preextraction... | Rockefeller University Press

Figure 1.

$Figure 1. Coupling RNase preextraction with high-resolution microscopy allows detection of NHEJ proteins at laser-induced DNA damage. (A) Localizations of Ku80 and nucleolin were analyzed in undamaged U2OS cells by immunofluorescence without CSK (no CSK), with preextraction using CSK only (CSK), or CSK combined with RNase A (CSK+R). (B) Analysis by immunoblotting of NHEJ proteins in whole-cell extracts (WCE) and in fractions retained after CSK extraction (−R) or CSK+R extraction (+R). (C) U2OS cells were microirradiated, postincubated for 5 min, and fixed without CSK or with preextraction using CSK or CSK+R. (D) Analysis by immunoblotting of expression levels of GFP-FLAG-Ku70 and GFP-FLAG-Ku80 in U2OS stable cells using antibodies against Ku70 and Ku80. (E) U2OS cells stably expressing GFP-FLAG-Ku70 (top) or GFP-FLAG-Ku80 (bottom) were microirradiated, postincubated for 5 min, and preextracted with CSK+R. Immunofluorescence was performed with an anti-GFP antibody. (F) Analysis by immunoblotting of GFP-FLAG-XRCC4 expression levels in U2OS stable cells with an antibody against XRCC4. (G) U2OS cells stably expressing GFP-FLAG-XRCC4 (clone 01) were microirradiated, postincubated for 5 min, and fixed without preextraction (top, no CSK) or preextracted with CSK+R (middle and bottom rows, CSK+R). In this figure, insets represent twofold zoom to highlight microfoci (boxed regions) formed by NHEJ proteins. The position of each nucleus, as defined by DAPI staining, is highlighted by a dotted line. Bars: (white) 10 µm; (green) 1 µm.$

Coupling RNase preextraction with high-resolution microscopy allows detection of NHEJ proteins at laser-induced DNA damage. (A) Localizations of Ku80 and nucleolin were analyzed in undamaged U2OS cells by immunofluorescence without CSK (no CSK), with preextraction using CSK only (CSK), or CSK combined with RNase A (CSK+R). (B) Analysis by immunoblotting of NHEJ proteins in whole-cell extracts (WCE) and in fractions retained after CSK extraction (−R) or CSK+R extraction (+R). (C) U2OS cells were microirradiated, postincubated for 5 min, and fixed without CSK or with preextraction using CSK or CSK+R. (D) Analysis by immunoblotting of expression levels of GFP-FLAG-Ku70 and GFP-FLAG-Ku80 in U2OS stable cells using antibodies against Ku70 and Ku80. (E) U2OS cells stably expressing GFP-FLAG-Ku70 (top) or GFP-FLAG-Ku80 (bottom) were microirradiated, postincubated for 5 min, and preextracted with CSK+R. Immunofluorescence was performed with an anti-GFP antibody. (F) Analysis by immunoblotting of GFP-FLAG-XRCC4 expression levels in U2OS stable cells with an antibody against XRCC4. (G) U2OS cells stably expressing GFP-FLAG-XRCC4 (clone 01) were microirradiated, postincubated for 5 min, and fixed without preextraction (top, no CSK) or preextracted with CSK+R (middle and bottom rows, CSK+R). In this figure, insets represent twofold zoom to highlight microfoci (boxed regions) formed by NHEJ proteins. The position of each nucleus, as defined by DAPI staining, is highlighted by a dotted line. Bars: (white) 10 µm; (green) 1 µm.