Cells were collected, washed and resuspended in 0.375 volumes of PBS
and mixed with 0.375 volumes of sample buffer (0.1M Tris pH 6.8, 4% SDS, 12%
β-mercaptoethanol). Subsequently, 4x NuPage LDS sample buffer (Thermo
Fisher Scientific) was added and samples were mixed by vortexing for 15 s
and boiled at 95 °C for 10 min. A second pulse of vortexing and
boiling was performed prior to subjecting equivalent protein amounts to gel
electrophoresis and proteins were transferred onto nitrocellulose membranes.
Proteins were detected using the appropriate primary and HRP-conjugated
secondary antibodies at a 1:10,000 dilution. Primary antibodies used in this
study include mouse anti-FLAG (Sigma-Aldrich F1804, 1:2,000), mouse anti-HA
(Sigma-Aldrich H3663, 1:5,000), rabbit anti-USP28 (Bethyl Laboratories
A300–898A, 1:1,000), mouse anti-p53 (DO-1, Santa Cruz Biotechnology
sc-126, 1:5,000), sheep anti-TRAIP (a gift from Niels Mailand, 1:500),
rabbit anti-phosphoRPA2S4/8 (Bethyl Laboratories
A300–083A, 1:10,000), rabbit anti-RPA2 (Bethyl Laboratories
A300–244A, 1:5,000) rabbit anti-γ-H2AX (Bethyl Laboratories
A300–081A, 1:10,000), rabbit H2AX (Bethyl Laboratories
A300–081A, 1:5,000), rabbit anti-ATM (Cell signaling #2873, 1:1,000),
rabbit anti-phosphoCHK2T68 (Cell signaling #2197, 1:1,000), mouse
anti-CHK2 (Cell signaling #3440, 1:500), and mouse anti-vinculin
(Sigma-Aldrich V9131, 1:25,000).
and mixed with 0.375 volumes of sample buffer (0.1M Tris pH 6.8, 4% SDS, 12%
β-mercaptoethanol). Subsequently, 4x NuPage LDS sample buffer (Thermo
Fisher Scientific) was added and samples were mixed by vortexing for 15 s
and boiled at 95 °C for 10 min. A second pulse of vortexing and
boiling was performed prior to subjecting equivalent protein amounts to gel
electrophoresis and proteins were transferred onto nitrocellulose membranes.
Proteins were detected using the appropriate primary and HRP-conjugated
secondary antibodies at a 1:10,000 dilution. Primary antibodies used in this
study include mouse anti-FLAG (Sigma-Aldrich F1804, 1:2,000), mouse anti-HA
(Sigma-Aldrich H3663, 1:5,000), rabbit anti-USP28 (Bethyl Laboratories
A300–898A, 1:1,000), mouse anti-p53 (DO-1, Santa Cruz Biotechnology
sc-126, 1:5,000), sheep anti-TRAIP (a gift from Niels Mailand, 1:500),
rabbit anti-phosphoRPA2S4/8 (Bethyl Laboratories
A300–083A, 1:10,000), rabbit anti-RPA2 (Bethyl Laboratories
A300–244A, 1:5,000) rabbit anti-γ-H2AX (Bethyl Laboratories
A300–081A, 1:10,000), rabbit H2AX (Bethyl Laboratories
A300–081A, 1:5,000), rabbit anti-ATM (Cell signaling #2873, 1:1,000),
rabbit anti-phosphoCHK2T68 (Cell signaling #2197, 1:1,000), mouse
anti-CHK2 (Cell signaling #3440, 1:500), and mouse anti-vinculin
(Sigma-Aldrich V9131, 1:25,000).