Figure 2.

Glo1 Y136 is phosphorylated by multiple different kinases including all members of the Src family.(A) Schematic diagram illustrating the tyrosine kinase CRISPR-Cas9 screen. A library of sgRNAs was generated to target each of the 90 known tyrosine kinases. HeLa cells were transfected with two sgRNAs for each kinase simultaneously. After puromycin selection, immunoblots were performed on lysates from each well using the pGlo1(Y136) antibody. (B) Knockdown of several different kinases in HeLa cells leads to a partial reduction in Glo1 Y136 phosphorylation. Tyrosine kinase knockouts were performed as in panel A, and immunoblots were done on the transiently-selected cell pools. Knockout of all tyrosine kinases is shown in Suppl. Figure 2. Shown here are two biological replicates of the promising candidates (47 of the 90 kinases) which showed a reduction in pGlo1(Y136) in the main screen. Knockouts causing a drop in the pGlo1/total Glo1 ratio in both biological replicates are indicated in blue. (C–D) Multiple different kinases can phosphorylate Glo1 Y136 in an in vitro kinase assay, including members of the Src family. (E) siRNA-mediated knockdown of Src kinase recapitulates results from the CRISPR-Cas9 screening (Suppl. Figure 3B), causing reduced Glo1(Y136) phosphorylation. A pool of 4 siRNAs was used to knock down either Src kinase or luciferase as negative control (“control”). n = 9, ∗∗∗p = 0.002 by 2-sided unpaired t-test. (F) Inhibition of the Src kinase family does not lead to a drop in Glo1 Y136 phosphorylation. HeLa cells were incubated for 30 min with either the broad range Src inhibitor Saracatinib and/or the Abl1 inhibitor Nilotinib. Inhibition of the Src kinase family was confirmed using pSrc(Y417) or pFak (Y925) antibodies.