Western Blot Analysis of PIP5K1γ Expression

Protein from cultured fibroblasts was harvested and lysates were blotted as previously described.^{111 (link)} anti-PIP5K1γ (ABS190; 1:300; Sigma) and anti-β-actin (MA1-91399; 1:1000; Thermo-Fisher) were applied to transferred membranes overnight at 4°C. Blot bands were detected by Sapphire Biomolecular Imager (Azure Biosystems) after 1 h incubation in the following secondary antibodies: goat anti-rabbit 680RD (P/N 926–68071, 1:10,000; LI-COR), goat anti-Mouse 800CW (P/N 925–32210, 1:10,000; LI-COR). Images were processed on ImageJ using the BioImporter plugin tool to calculate the protein expression for each band. Protein abundance was first normalized to beta-actin intensity then normalized to control cell intensity.