Iav m1

Cells were fixed with 2% paraformaldehyde and permeated with Triton X-100. Cellular proteins were determined by immunofluorescence using specific antibodies against GBF1 (BD Biosciences, San Jose, CA, USA), GM130 (Abcam, Cambridge, UK), and IAV M1 (Genetex, Irvine, CA, USA). Alexa 488 and 594 conjugated secondary antibodies (Thermo Fisher, Grand Island, NY, USA) were used to obtain co-stained fluorescence images. DAPI was used to stain the cell nucleus. The images were acquired either by Nikon microscope (Nikon Eclipse Ti Microscope, Tokyo, Japan) or by super-resolution microscopy (ELYRA S.1, Carl Zeiss, Thornwood, NY, USA). Images were acquired by a 63×/1.40 plan-apochromat or a 100×/1.46 alpha plan-APO oil immersion objective lens and post-processed using Zeiss ZEN Black version 2.3 (Carl Zeiss, White Plains, NY, USA) software. All imaging analyses were repeated at least three times, and major features were determined by two independent examiners in a blinded fashion. The degree of colocalization of GBF1 and M1 was determined by randomly sampling 10 fields in each individual sample and counting the percentage of cells showing co-staining. In total, 10 replicates were performed.

To test for coimmunoprecipitation, the precipitated proteins were separated by SDS-PAGE and immunoblotted using antibodies against GBF1 (Santa Cruz, CA, USA), IAV M1, NP, and PA (Genetex, Irvine, CA, USA). Active ARF1 pull down and detection kit (Thermofisher, Waltham, MA, USA) was used to quantify the amount of GTP bound ARF1 (ARF-GTP) based on the manufacturer’s protocol. Equal amounts of proteins were loaded for the pull-down.