Fig. 9 | Nature Communications

Fig. 9

From: Endothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis

Fig. 9

ADORA2A regulates HIF-1α protein level through activation of the PI3K/Akt and MEK/ERK-mediated translational machinery. a Western blot analysis of HIF-1α at protein level in HRMECs. Cells were transfected with siA2AR or the siCtrl for 48 h, and then treated with MG-132 (10 µM) for 1, 2 and 4 h. b Western blot analysis of HIF-1α at protein level in HRMECs. Ad-Ctrl and Ad-A2AR-infected HRMECs were first treated with CoCl2 (200 μM) for 8 h to increase HIF-1α protein level and then further treated with CHx (50 µM) for the indicated time periods. c, d The relative protein levels of HIF-1α were quantified by comparing the intensities of protein bands at the indicated times to that at time 0. n = 3, ***P < 0.001. Data are represented as means ± s.e.m. Statistical significance was determined by two-way ANOVA followed by Bonferroni test. e Western blot analysis of the levels of total-ERK (t-ERK), phospho-ERK1/2 (p-ERK1/2), phospho-p38 (p-p38), total-p38 (t-p38), phospho-JNK1/2 (p-JNK1/2), total-JNK1/2 (t-JNK1/2), phospho-p70S6K (p-p70S6K), total-p70S6K (t-p70S6K), phospho-eIF-4E (p-eIF-4E), and total-eIF-4E (t-eIF-4E) in HRMECs transfected with siA2AR or siCtrl, with or without adenosine treatment under hypoxia. n = 3. f Western blot analysis of HIF-1α and phosphoproteins in HRMECs infected with Ad-A2AR, with or without adenosine treatment. n = 3. g Western blot analysis of HIF-1α and phosphoproteins in HRMECs infected with Ad-Ctrl and Ad-A2AR in the presence of U0126 (10 μM) or LY294002 (10 μM). n = 4