Fig 1: Cell surface biotinylation reveals that HSPA1A’s PM embedding significantly decreases in the presence of biosensors masking PI(4)P and PI(3)P. Representative cropped Western blots showing the total and biotinylated fractions of HEK293 cell lysates transfected with (A) GFP-HSPA1A and eGFP (see Supplemental Figure S1 for complete blots), and GFP-HSPA1A and GFP-P4M-SidMx2, (B) HSPA1A-myc and eGFP (see Supplemental Figure S1 for complete blots), and HSPA1A-myc and GFP-EEA1. HSPA1A is shown in (A) at the anti-GFP panel and in (B) at the anti-myc panel. Native actin and ATP1A1 were used a cytosolic and membrane controls, respectively. P4M-SidMx2 is visible in (A) at the bottom anti-GFP panel and EEA1 at the anti-GFP panel of (B). M: molecular size marker (Fisher BioReagents™ EZ-Run™ Prestained Rec Protein Ladder; approximate sizes shown on the left side of the blots). (C) Quantification of the antibody detected signals of the HSPA1A (GFP or myc tagged) in the absence (graph columns one-two and five-six) or presence (graph columns three-four and seven-eight) of P4M-SidMx2 or EEA1 presented as a ratio between the biotinylated (PM) fraction and the total cell lysate. Densitometry values are averages of three independent experiments (n = 3). These values were normalized to controls (control set to 100%), and the standard deviation was scaled accordingly. Center lines show the medians; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; crosses represent sample means.