Anti pgp

Cells were lysed in 50 mM TrisHCl pH 8.0 containing 150 mM NaCl, 1 mM EDTA, 1% NP-40. Cell extracts (100 μg/lane) were resolved by 8 or 10% SDS-PAGE, transferred to nitrocellulose sheets at 100 mA for 16 h, and probed with anti-TRPM7 (Bethyl), anti-MagT1 (ProteinTech), anti-PgP (ThermoScientific), and anti-actin (Sigma Aldrich) antibodies. Secondary antibodies were labelled with horseradish peroxidase (GE Healthcare). The SuperSignal chemiluminescence kit (Pierce) was used to detect immunoreactive proteins. All the results were reproduced at least three times and a representative Western blot is shown. Densitometric analysis was performed by the ImageJ software and ratio between the protein of interest and actin was calculated on three separate experiments (see Supplementary).

At the time points indicated, CVB3 infected cells were imaged using a Nikon Ti2 inverted epifluorescence microscope equipped with a Qi2 camera (Nikon, Tokyo, Japan) using NiS Elements software version AR.5.30.05. Cells were fixed in ice-cold methanol and stained with anti-PECAM-1 (CD-31) (Cat# PA5-16301 Thermo Fisher), anti-VE-Cadherin (Cat# sc-52752 [BV9] Santa Cruz), anti-Glut1 (Cat# MA5-11315 Thermo Fisher), anti-P-gp (Cat# MA5-13854 Thermo Fisher), anti-Occludin (Cat# OC-3F10, Invitrogen), anti-Claudin-5 (Cat# 35-2500, Invitrogen), and anti ZO-1 (Cat# 33-9100 Invitrogen) as previously described (Stebbins et al., 2016 (link); Espinal et al., 2022b (link)) and imaged on the Nikon Ti2 microscope. Images were analyzed with NIH ImageJ software (FIJI).