Journal: Journal of Virology

Article Title: Marine medaka PKCα promotes red-spotted grouper nervous necrosis virus entry by orchestrating MYL3-mediated macropinocytosis and cofilin-dependent actin remodeling

doi: 10.1128/jvi.02064-25

Figure Lengend Snippet: MmPKCα facilitates RGNNV entry via macropinocytosis. ( A ) hMMES1 or MmMYL3-overexpressing hMMES1 cells were treated with PBS or Go 6983 or transfected with siMmPKCα, and then infected with RGNNV (MOI = 1) in medium containing Alexa Fluor 647-conjugated dextran (10,000 MW). At 4 hpi, cells were fixed, and nuclei counterstained with DAPI and imaged on a confocal microscope. Bar = 10 µm. ( B ) hMMES1 cells were transfected with MmMYL3-Flag plasmids for 24 h and then were subjected to immunoblot assays using anti-PKCα or anti-PKCα (phosphor T497) abs. ( C ) MmPKCα-Flag and MmMYL3-Myc plasmids were transfected into HEK293T cells as indicated for immunofluorescence analysis by using anti-Flag (green) or anti-Myc (red) abs. Nuclei were stained with DAPI. Bar = 10 µm. ( D ) IP (with anti-Flag abs) and immunoblot analysis (with anti-Flag and anti-Myc abs) of HEK293T cells transfected with plasmids encoding MmMYL3-Flag and MmPKCα-Myc for 48 h. ( E ) The lysates of HEK293T cells transfected with the indicated plasmids were pulled down with purified MmMYL3-GST or GST proteins. The proteins bound to MmPKCα, and the inputs were immunoblotted with anti-GST and anti-Flag abs.The results are presented as mean ± SD. Statistical significance was determined by an unpaired two-tailed Student’s t test. * P < 0.05, ** P < 0.01. Data are representative of three independent experiments.

Article Snippet: Additionally, donkey anti-mouse or goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary abs, as well as Alexa Fluor 555 and Alexa Fluor 488, Alexa Fluor 647-dextran (10,000 MW), were obtained from Invitrogen (Carlsbad, CA, USA). iFluorTM 488 (40736ES75) phalloidin was purchased from Yeasen, while magnetic beads of anti-Flag (HY-K0207), anti-c-Myc (HY-K0206), and anti-GST (HY-K0222) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Transfection, Infection, Microscopy, Western Blot, Immunofluorescence, Staining, Purification, Two Tailed Test