Fig. 5

Phosphorylation status of signaling complexes in SUSD4-expressing cells favors autophagy initiation. A Comparison in phosphorylation status of a wide range of proteins in SUSD4-expressing BT-20 cells and mock control cells using the Human Phospho-Kinase Antibody Array (n = 2 technical repeats). B-E Phospho-AMPKα1 levels in SUSD4-expressing BT-20 cells or MDA-MB-468 cells and corresponding mock control cells treated with AICAR (B and D) or A-769662 (C and E). Representative immunoblots (B and C) and densitometric analysis (D and E) of phospho-AMPKα1 levels in both treated and untreated cells (n = 3 biological repeats for treatment with A-769662 and BT-20 cells treated with AICAR, n = 4 biological repeats for MDA-MB-468 cells treated with AICAR). F and G Representative immunoblots (F) and densitometric analysis (G) showing the phosphorylation status of ULK1 at three distinct phosphorylation sites in BT-20 and MDA-MB-468 cells expressing SUSD4 and corresponding mock control cells (n = 4 biological repeats for S757 and S555 in BT-20 cells, n = 3 biological repeats for S317 in BT-20 cells and all phosphorylation sites in MDA-MB-468 cells). H and I Representative immunoblot (H) and densitometric analysis (I) of phospho-activated Beclin-1 in BT-20 cells or MDA-MB-468 cells expressing SUSD4 compared to the respective mock control cells. J and K Representative immunoblot (J) and densitometric analysis (K) of phospho-Atg14 levels in BT-20 cells or MDA-MB-468 cells expressing SUSD4 and corresponding mock control cells. L-N Representative immunoblots and densitometric analyses showing phospho-LKB1 levels in BT-20 SUSD4 cells and mock control cells (L), BT-20 EGFR knockout cells expressing or lacking SUSD4 (M), and MDA-MB-468 cells expressing or lacking SUSD4. All results were repeated in at least three independent biological experiments. The p-value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001