Fig 2: HSPA1A’s plasma membrane (PM) localization significantly increases during recovery from mild heat shock.(A) Quantification of confocal imaging data showing the corrected total cell fluorescence (CTCF) ratio of HSPA1A fluorescence at the PM to the rest of the cell. Localization was analyzed in cells under control conditions (37°C) or after heat shock (1 h at 42°C), followed by recovery at 37°C for 0–24 h. Data represent three independent experiments, with individual cells shown as open circles (total N=30). The center lines show medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range; crosses represent sample means. Statistical significance was determined using one-way ANOVA (Analysis of Variance) followed by a post-hoc Tukey HSD (Honestly Significant Difference) test. (B) Cell surface biotinylation confirms that HSPA1A’s PM association significantly increases in response to heat shock, mirroring the confocal imaging results. Representative cropped. Western blots show total and biotinylated fractions from HEK293 cell lysates expressing HSPA1A-myc (complete blots in Supplemental Figure 1). Actin and ATP1A1 were used as cytosolic and membrane controls, respectively. M: molecular size marker (Fisher BioReagents™ EZ-Run™ Prestained Rec Protein Ladder; approximate sizes indicated on the left).

Fig 3: HSPA1A's plasma membrane (PM) localization significantly increases during recovery from mild heat shock. (a) Confocal microscopy was used to assess total membrane-associated GFP-HSPA1A using corrected total cell fluorescence (CTCF), which captures both peripherally associated and embedded protein pools at or near the PM. Localization was analyzed in cells under control conditions (37 °C) or after heat shock (1 h at 42 °C), followed by recovery at 37 °C for 0–24 h. Data represent three independent experiments, with individual cells shown as open circles (total N = 30). The center lines show medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range; crosses represent sample means. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by a post-hoc Tukey honestly significant difference (HSD) test. (b) Cell surface biotinylation, which selectively labels HSPA1A exposed on the extracellular surface of the PM, provides biochemical confirmation of membrane surface localization. The increase in biotinylated HSPA1A following heat shock mirrors the confocal imaging data, supporting robust PM association. Representative cropped Western blots show total and biotinylated fractions from HEK293 cell lysates expressing HSPA1A-myc (complete blots in Supplemental Figure 2). Actin and ATP1A1 were used as cytosolic and membrane controls, respectively. (c) Endogenous HSPA1A localizes to the PM during recovery from heat shock, as determined by detergent-free PM protein enrichment and Western blot analysis. HEK293 cells were subjected to heat shock (1 h at 42 °C) and allowed to recover for 0–24 h. These biochemical results support and extend the findings from confocal imaging and biotinylation assays. Cytosolic and PM-enriched fractions were probed for HSPA1A. ATP1A1 and GAPDH were used as controls for PM and cytosolic fractions, respectively (complete blots in Supplemental Figure 2). M: molecular size marker (Fisher BioReagents™ EZ-Run™ Prestained Rec Protein Ladder; approximate sizes indicated on the left).