Fig. 3.

Talin is SUMOylated in isolated focal adhesions and is cleaved in concentrated isolated FAs. 3A. Western blotting showed that the main FA proteins were present in the isolated FAs as well as in the whole cell lysates as talin, vinculin, FAK and actin. 3B. The isolated FAs were used in the SUMO binding assay which showed that talin was a substrate for SUMOylation at 250 kDa. Additionally, talin seemed to be cleaved. The 220 kDa fragments of talin were found in the eluted fraction but not in the unbound whole cell lysate fraction. The fragmented talin could be SUMOylated (n = 3, individual replicated experiment). After 1 h of 100 µM GA treatment, the band intensity for the SUMOylated talin was significantly reduced (n = 7, mean ± SEM, p = 0.036, two-tailed unpaired t-test, individual replicated experiment, data was combined from the HA-SUMO-2 IP pulldown and the SUMO binding assay with FAs). 3C. GAPDH was not detected as SUMOylated using the SUMO binding assay (n = 3, individual replicated experiment). 3D. The cells were incubated with the SUMO matrix-containing columns in the SUMO binding assay to obtain eluted SUMOylated proteins. These were reserved in 0.1% formic acid previously and sent for mass spectrometry LC-MS/MS analysis. Talin, filamin A, actin and vinculin were detected as SUMOylation substrates.