Sample buffer solution

The harvested cells were washed with PBS, suspended in RIPA Buffer (Nacalai tesque, catalog no. 16488-34) containing Protease Inhibitor Cocktail (Nacalai tesque, catalog no. 25955-24), incubated on ice for 15 minutes, and centrifuged (12,000 × g, 10 minutes, 4°C). The supernatant was collected as a cell lysate. Next, we added Sample Buffer Solution (Nacalai tesque, catalog no. 09499-14) containing 100 mmol/L Dithiothreitol (DTT) prior to boiling, and ran the samples on 4%–20% gradient gels (TEFCO, catalog no. 01-022-10) for SDS-PAGE, and performed transfer on Immobilon-P PVDF membranes (Millipore, catalog no. IPVH00010). Blocking with 5% skim milk in TBS-T for 1 hour at room temperature. The membranes were incubated with 0.5 μg/mL anti-CAPRIN-1 antibodies, 0.3 μg/mL anti-GAPDH antibody (Abcam, catalog no. ab9485, RRID:AB_307275), 0.1 μg/mL anti-Sodium Potassium ATPase antibody (Abcam, catalog no. ab76020, RRID:AB_1310695) in 5% skim milk/TBS-T for 1 hour at room temperature, after washing the membrane four times with TBS-T, incubated with the second antibody, that is, HRP anti-rabbit IgG, HRP anti-mouse IgG, and HRP anti-human IgG for 1 hour at room temperature. Signals were detected using Western Lightning Plus-ECL (PerkinElmer, catalog no. NEL104001EA) on a FUSION SOLO.6S.EDGE (Vilber Lourmat) chemiluminescence imaging system.

Whole proteins
of cells were extracted
with sample buffer solution (Nacalai Tesque, Kyoto, Japan), incubated
at 95 °C for 5 min, and then centrifuged at 15,000 rpm for 5
min at 4 °C. Supernatants were subsequently separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with
a gradient gel (Atto, Tokyo, Japan), followed by electrophoretic transfer
onto PVDF membrane (Merck Millipore). After the blotting, the membranes
were blocked in Blocking One (Nacalai Tesque) for 45 min and then
incubated with primary antibodies [anti-GFAP mouse monoclonal antibody
(1:2500, Merck Millipore) and anti-ACTB mouse monoclonal antibody
(1:2500, Santa Cruz, Dallas, TX)] at 4 °C overnight, followed
by incubation at room temperature for 2 h with HRP-conjugated secondary
antibodies (Santa Cruz). The Can Get Signal Immunoreaction Enhancer
Solution Kit (Toyobo) was used as an antibody diluent for the signal
enhancement. Signal was detected with LAS-4000 (GE Healthcare, Chicago,
IL) using Chemi-Lumi One L (Nacalai Tesque), and signal intensities
were calculated with ImageQuant TL Software (GE Healthcare).

Cells were lysed in Sample Buffer Solution (Nacalai tesque, Kyoto, Japan), heated at 95℃ for 10 min, and subjected to sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred from the gels onto Immobilon-P PVDF membranes (Millipore, Burlington, MA, USA). ACE2 and β-actin protein were detected on blots by probing with anti-hACE2 antibody (#4355, Cell Signaling Technologies, Danvers, MA, USA) and anti-actin antibody (MAB1501, Chemicon, Temecula, CA, USA), respectively. Immune complexes were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies and SuperSignal West Femto Maximum Sensitivity Substrate (Pierce; Thermo Fisher Scientific).