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. 2016 Apr 4;17(5):671–681. doi: 10.15252/embr.201541455

Figure 1. PALB2 phosphorylation is induced upon DNA damage in an ATM/ATR‐dependent manner.

Figure 1

  1. U2OS cells were harvested at the indicated time points after treatment with ionizing radiation (15 Gy) followed by analysis by SDS–PAGE and immunoblotting with antibodies recognizing PALB2 and vinculin (NT, no treatment). The lysate from the 24 h time point was treated with phosphatase (Ppase).
  2. U2OS cells were left untreated or treated with ATM (KU 55933, 10 μM), ATR (ETP‐464, 1 μM), or DNA‐PK (NU7441, 1 μM) inhibitors 30 min prior to exposure to IR (15 Gy, 2 h recovery). Immunoblots were performed with the indicated antibodies.
  3. U2OS cells were left untreated (NT) or exposed to HU (2 mM) for the indicated times. The lysate from the 24 h time point was treated with phosphatase (Ppase). Immunoblots were performed with the indicated antibodies.
  4. Untreated U2OS cells or cells exposed to either HU (2 mM, 24 h) or IR (15 Gy, 2 h recovery) alone or together with ATM inhibitor (KU55933, 10 μM) or inhibitors to ATR (ATR#1: ETP‐464, 1 μM; ATR#2: AZ‐20, 3 μM) were harvested and lysates were prepared. The inhibitors were added 30 min prior to HU or IR treatment. Immunoblots were performed with the indicated antibodies.
  5. ATR kinase assay was performed with N‐terminal PALB2 (aa 1‐560; GST–PALB2–N (Fig 2A)) as a substrate and HA‐tagged ATR as kinase and visualized by Fujifilm Phosphroimager. 293T cells transfected with empty vector (EV), GST, and ATR inhibition (AZ‐20, 30 μM) are controls. HA‐ATR and GST–PALB2–N were controlled by immunoblotting with HA and GST antibodies.