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. 2020 Mar 18;39(10):e103111. doi: 10.15252/embj.2019103111

Figure 2. Oxidative stress enhances CHK2‐Beclin 1 interaction.

Figure 2

  • A
    Immunoprecipitation assays testing the physical interaction between CHK2 and proteins encoded by autophagy‐related genes in HCT116 cells. Lysates were extracted for immunoprecipitation with CHK2‐specific antibody or control IgG, followed by probing with antibodies specific for Ulk1, Atg5, Beclin 1, Atg7, or LC3.
  • B
    Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t = 0) and after HBSS starvation in the cytoplasmic fractions.
  • C
    Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t = 0) and after H2O2 (500 μM) stimulation in the cytoplasmic fractions.
  • D
    HCT116 cells were pretreated with NAC in RPMI 1640 complete medium for 4 h and then cultured for 1 h in HBSS starvation. The cytoplasmic lysates were subjected to immunoprecipitation with anti‐Beclin 1 antibody followed by immunoblotting with anti‐CHK2 and anti‐Beclin 1 antibodies. The expression of p‐CHK2 Thr68, CHK2, and Beclin 1 was monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.
  • E, F
    HCT116 cells were pretreated with CHK2 inhibitor II in RPMI 1640 complete medium for 4 h and then cultured for 1 h in HBSS starvation (E) or H2O2 (500 μM) stimulation (F). The cytoplasmic lysates were subjected to immunoprecipitation with anti‐Beclin 1 antibody followed by immunoblotting with anti‐CHK2 and anti‐Beclin 1 antibodies. The expression of p‐CHK2 Thr68, CHK2, and Beclin 1 was monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.
  • G–I
    HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 h post‐transfection, cells were treated or untreated (G) with HBSS starvation (H) or H2O2 (500 μM) stimulation (I) for 1 h and then collected for immunoprecipitation and Western blotting analysis. Immunoprecipitation was performed using anti‐Beclin 1 antibody followed by immunoblotting with anti‐Flag or anti‐Beclin 1 antibody.
  • J
    Western blot detection of p62 and LC3 in H1299 cells transfected with indicated plasmid in normal medium or after H2O2 (500 μM) cultured for 3 h.
  • K
    H1299 cells were pretreated with CHK2 inhibitor II in complete medium for 4 h and then cultured for 3 h in H2O2 (500 μM) stimulation. Western blot detection of p‐CHK2 Thr68, CHK2, p62, and LC3.

Source data are available online for this figure.