Phosphate buffered saline (pbs)

N1 and N2 were each diluted either in PBS (Quality Biological, Gaithersburg, MD) or DPBS (Thermo Fisher Scientific, Waltham, MA). The recipe for PBS, as provided by the manufacturer, is sodium phosphate dibasic (795 mg/l, 5.6 mM), potassium phosphate monobasic (1.06 mM), and sodium chloride (9000 mg/l, 154 mM). The recipe for DPBS, as provided by the manufacturer, is calcium chloride (100 mg/l, 0.9 mM), magnesium chloride (100 mg/l, 0.49 mM), potassium chloride (200 mg/l, 2.66 mM), potassium phosphate monobasic (200 mg/l, 1.47 mM), sodium chloride (8000 mg/l, 137.93 mM), and sodium phosphate dibasic (2160 mg/l, 8.06 mM). Samples were stored at −80 °C and at 4 °C for a total of eight different sets of combinations of buffer and temperature conditions. NA stored at −80 °C was thawed at each timepoint and then returned to frozen storage until the next timepoint. NA activity was tested at weeks 0, 4, 12, 24, and 36. NA in PBS at week 36 was subsequently “rescued” through dilution in DPBS to compare its activity to NA in PBS at 36 weeks and NA in DPBS at 36 weeks. The stability experiment was also repeated with N1 in PBS, N1 in DPBS, N2 in PBS, and N2 in DPBS at RT and activity was tested at weeks 0, 3, 8, 12, and 24.

HEK293T cells (ATCC: The Global Bioresource Center) were cultured in 35-mm petri dishes in DMEM (Corning Cellgro, 15-013-CV) supplemented with 10% FBS (Sigma-Aldrich) and 1%GlutaMax (Cellutron Life Technologies) under 5% CO₂ at 37 °C. Cells were transiently transfected with the plasmid DNA of interest using FuGene HD following manufacturer’s instruction (Promega). Equal amount of plasmid DNA of KCNQ1 (or KCNQ1-GFP or its mutants) and KCNE1 were used. Confocal images and current recordings were conducted 48–56 h after transient expression of the channels.
HEK293T cells were transfected with GFP-tagged KCNQ1 (1.5 µg) and KCNE1(1.5 µg). 6 h after transfection, cells were plated on glass bottom dishes (MatTek Corporation). Forty-eight hours after transfection, cells were washed two times with PBS (Quality Biological) and incubated at 37 °C for treatment in extracellular recording solution (below). For all treatments, cells were incubated at 37 °C, 5% CO₂. DMSO treated cells were used as control for PAO (2 μM) and WMN (0.01 μM, 5 μM) treatment. Rapamycin (1 μM) was applied for 30 min to induce PJ, PJ-SAC, PJ-INPP5E, or PJ-DEAD translocation to the plasma membrane. Confocal images and patch clamp experiments were performed at room temperature immediately after treatment.

SARS-CoV-2 lung titers were quantified by homogenizing harvested lungs in PBS (Quality Biological Inc.) using 1.0 mm glass beads (Sigma Aldrich) and a Beadruptor (Omini International Inc.). Homogenates were added to near-confluent Vero E6 cultures and SARS-CoV-2 virus titers determined by counting plaque forming units using a 6-point dilution curve.