Immunofluorescence and Immunoblotting Analysis

Immunofluorescence assays were performed as described previously [9 (link)]. Primary antibodies against p-H2A.X (Ser139), and p-53BP1 (Ser1778) (Cell Signaling Technology), were diluted 1:250 with PBS with 0.1% Tween-20 (PBST), and incubated overnight at 4° C. Secondary antibodies were either Alexa Fluor 594-conjugated secondary (Invitrogen), DyLight^TM 488 (Rockland) or DyLight™ 549, diluted 1:1000 in PBST. Slides were mounted with Prolong Gold (Invitrogen). Cellular lysates were produced and analyzed by immunoblotting as described previously [9 (link)]. Primary antibodies were against p-H2A.X (Ser139), H2A.X, p-53BP1 (Ser1778), 53BP1, CHK1 (Cell Signaling Technology) p-CHK1 (Ser345, R&D Systems) and GAPDH (Santa Cruz). Results were quantified using ImageJ and Image Lab from BioRad.

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Colavito S.A., Platt J.T., Held M.A., Liu Z., Sokup R, & Stern D.F. (2019). Combinatorial drug screening of mammary cells with induced mesenchymal transformation to identify drug combinations for triple-negative breast cancer. Oncotarget, 10(47), 4822-4839.

Publication 2019

p-H2A.X DyLight™ 549 Prolong Gold GAPDH ImageJ Image Lab

53bp1 Alexa 594 Antibodies Cellular Gapdh Gold H2a x Immunofluorescence assays Tween 20

Corresponding Organization : University of Wisconsin–La Crosse

Other organizations : Yale Cancer Center, University of New Hampshire, Yale University

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Variable analysis

independent variables

Primary antibodies against p-H2A.X (Ser139), and p-53BP1 (Ser1778) (Cell Signaling Technology)

dependent variables

Cellular localization and expression of p-H2A.X (Ser139) and p-53BP1 (Ser1778) proteins (as measured by immunofluorescence assays)
Cellular expression of p-H2A.X (Ser139), H2A.X, p-53BP1 (Ser1778), 53BP1, CHK1, p-CHK1 (Ser345) (as measured by immunoblotting)

control variables

Dilution of primary antibodies (1:250 with PBS with 0.1% Tween-20)
Incubation time of primary antibodies (overnight at 4°C)
Dilution of secondary antibodies (1:1000 in PBST)
Mounting medium (Prolong Gold)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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