Journal: Pharmaceuticals

Article Title: Tyrphostin AG1024 Suppresses Coronaviral Replication by Downregulating JAK1 via an IR/IGF-1R Independent Proteolysis Mediated by Ndfip1/2_NEDD4-like E3 Ligase Itch

doi: 10.3390/ph15020241

Figure Lengend Snippet: TGEV infection did not activate IR/IGF-1R signaling but induced JAK1 activation in the ST cell. ST cells were infected by TGEV at an MOI of 7 or respectively stimulated with IGF-1 (10 nM) ( A ) or insulin (100 nM) ( B ). The resulting cell lysates, harvested at the indicated time points of 0.5 h, 1 h, 3 h, and 6 h, were subjected to western analyses with antibodies against p-JAK1, JAK1, p-IGF-1R, IGF-1R, p-insulin R, insulin R or the internal loading controls vinculin or β-actin, respectively. *, p < 0.05; **, p < 0.01. Results shown are averages ± SD from three independent experiments.

Article Snippet: Western blotting was performed as described [ , ] with antibodies against β-actin (1:2000, catalog # 4970), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000, catalog # 2118), p-IGF-I Rβ (Tyr1135/1136)/insulin Rβ (Tyr1150/1151) (1:1000, catalog # 3024), IGF-I Rβ (1:1000, catalog # 3027), insulin Rβ (1:1000, catalog # 3025), p-JAK1 (Y1022/1023) (1:1000, catalog # 3331), and JAK1 (1:1000, catalog # 3344) from Cell Signaling Technology, Danvers, MA, USA; vinculin (GTX109749, 1:2000) (GeneTex, Irvine, CA, USA); OC43 N protein (MAB9013, 1:1000) (Merck, La Jolla, CA, USA) and an antibody against TGEV N protein, as described [ ].

Techniques: Infection, Activation Assay, Western Blot