Journal: Biomolecules

Article Title: Phosphatidylinositol Monophosphates Regulate the Membrane Localization of HSPA1A, a Stress-Inducible 70-kDa Heat Shock Protein

doi: 10.3390/biom12060856

Figure Lengend Snippet: HSPA1A’s PM localization significantly decreases when PI(4)P and PI(3)P are masked by the biosensors P4M-SidMx2 and EEA1. ( A ) Representative images of HeLa cells expressing GFP-HSPA1A stained with WGA-FA555 PM stain and DAPI nucleus stain (top panels). Representative images of the co-expression of RFP-HSPA1A with GFP-P4M-SidMx2 (middle panels) and RFP-HSPA1A with GFP-EEA1 (bottom panels). The localization of HSPA1A was documented for all transfections at either control conditions (37 °C; top row of each panel) or heat-shocked conditions (1 h at 42 °C followed by 8 h at 37 °C; bottom row of each panel). Scale bar = 10 μm. ( B ) Quantification of the corrected total cell fluorescence (CTCF) as a ratio between the total HSPA1A fluorescence of the PM and the rest of the cell at control conditions and after heat shock in the presence or absence of lipid biosensors. The experiment was repeated three times, and the total number of cells (shown as open circles) per condition are shown at the bottom of the graph. The center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; crosses represent sample means.

Article Snippet: The antibodies used were: the OmicsLinkTM Anti-GFP Tag Antibody Mouse Monoclonal IgG1 [(CGAB-GFP-0050, Genecopoeia, Rockville, MD, USA); 1:1000- detects a protein band of approximately 30 kDa (empty GFP), two bands of approximately 98 kDa (GFP-HSPA1A and GFP-EEA1), several bands around 52 kDa of the remaining biosensor constructs (Lact-C2-GFP, GFP-P4M-SidMx2, GFP-PLCδ-PH)]; the Anti-c-Myc Antibody (9E10, Genecopoeia, Rockville, MD, USA) [monoclonal IgG (Dilution 1:1000)- detects a protein of approximately 74 kDa (HSPA1A-myc)]; the anti-HSP70 Monoclonal Antibody [(mouse IgG) (Dilution 1:1000) clone #C92F3A-5 (Enzo Life Sciences, Farmingdale, NY, USA; detects a protein around 74 kDa; native and overexpressed HSPA1A)]; THETM beta actin antibody, GenScript [Piscataway, NJ, USA (mouse mAb; A00702) 1:1000- detects a protein band of approximately 42 kDa; cytosolic loading control]; and the Na+/K+ ATPase α (ATP1A1) antibody RabMAb ® [(EP1845Y); (2047-1), Epitomics, Burlingame, CA, USA; 1:1000- detects a protein band of approximately 112 kDa plasma membrane loading control].

Techniques: Expressing, Staining, Transfection, Control, Fluorescence, Software

Journal: Biomolecules

Article Title: Phosphatidylinositol Monophosphates Regulate the Membrane Localization of HSPA1A, a Stress-Inducible 70-kDa Heat Shock Protein

doi: 10.3390/biom12060856

Figure Lengend Snippet: 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 for complete blots), and GFP-HSPA1A and GFP-P4M-SidMx2, ( B ) HSPA1A-myc and eGFP (see 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.

Article Snippet: The antibodies used were: the OmicsLinkTM Anti-GFP Tag Antibody Mouse Monoclonal IgG1 [(CGAB-GFP-0050, Genecopoeia, Rockville, MD, USA); 1:1000- detects a protein band of approximately 30 kDa (empty GFP), two bands of approximately 98 kDa (GFP-HSPA1A and GFP-EEA1), several bands around 52 kDa of the remaining biosensor constructs (Lact-C2-GFP, GFP-P4M-SidMx2, GFP-PLCδ-PH)]; the Anti-c-Myc Antibody (9E10, Genecopoeia, Rockville, MD, USA) [monoclonal IgG (Dilution 1:1000)- detects a protein of approximately 74 kDa (HSPA1A-myc)]; the anti-HSP70 Monoclonal Antibody [(mouse IgG) (Dilution 1:1000) clone #C92F3A-5 (Enzo Life Sciences, Farmingdale, NY, USA; detects a protein around 74 kDa; native and overexpressed HSPA1A)]; THETM beta actin antibody, GenScript [Piscataway, NJ, USA (mouse mAb; A00702) 1:1000- detects a protein band of approximately 42 kDa; cytosolic loading control]; and the Na+/K+ ATPase α (ATP1A1) antibody RabMAb ® [(EP1845Y); (2047-1), Epitomics, Burlingame, CA, USA; 1:1000- detects a protein band of approximately 112 kDa plasma membrane loading control].

Techniques: Western Blot, Transfection, Membrane, Marker, Control, Standard Deviation

Journal: Biomolecules

Article Title: Phosphatidylinositol Monophosphates Regulate the Membrane Localization of HSPA1A, a Stress-Inducible 70-kDa Heat Shock Protein

doi: 10.3390/biom12060856

Figure Lengend Snippet: Plasma membrane localization of HSPA1A is not affected by ionomycin. ( A ) Representative images of HeLa cells expressing GFP-HSPA1A (two top panels) and GFP-PLCδ (two bottom panel). Cells were either incubated with DMSO (untreated controls; first row of each panel) or treated with 10 µM ionomycin for 2 min (second row of each panel). Only heat-shocked cells are shown. The PM of all cells was stained with WGA-FA555 and the nucleus with DAPI (second and third picture of each panel, respectively). Scale bar = 10 μM. ( B ) Quantification of the corrected total cell fluorescence (CTCF) as a ratio between the total fluorescence at the PM and the rest of the cell at control conditions and after heat shock in the presence or absence of ionomycin. The experiment was repeated three times, and the total number of cells (shown as open circles) per condition are shown at the bottom of the graph. The center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; crosses represent sample means.

Article Snippet: The antibodies used were: the OmicsLinkTM Anti-GFP Tag Antibody Mouse Monoclonal IgG1 [(CGAB-GFP-0050, Genecopoeia, Rockville, MD, USA); 1:1000- detects a protein band of approximately 30 kDa (empty GFP), two bands of approximately 98 kDa (GFP-HSPA1A and GFP-EEA1), several bands around 52 kDa of the remaining biosensor constructs (Lact-C2-GFP, GFP-P4M-SidMx2, GFP-PLCδ-PH)]; the Anti-c-Myc Antibody (9E10, Genecopoeia, Rockville, MD, USA) [monoclonal IgG (Dilution 1:1000)- detects a protein of approximately 74 kDa (HSPA1A-myc)]; the anti-HSP70 Monoclonal Antibody [(mouse IgG) (Dilution 1:1000) clone #C92F3A-5 (Enzo Life Sciences, Farmingdale, NY, USA; detects a protein around 74 kDa; native and overexpressed HSPA1A)]; THETM beta actin antibody, GenScript [Piscataway, NJ, USA (mouse mAb; A00702) 1:1000- detects a protein band of approximately 42 kDa; cytosolic loading control]; and the Na+/K+ ATPase α (ATP1A1) antibody RabMAb ® [(EP1845Y); (2047-1), Epitomics, Burlingame, CA, USA; 1:1000- detects a protein band of approximately 112 kDa plasma membrane loading control].

Techniques: Clinical Proteomics, Membrane, Expressing, Incubation, Staining, Fluorescence, Control, Software

Journal: Biomolecules

Article Title: Phosphatidylinositol Monophosphates Regulate the Membrane Localization of HSPA1A, a Stress-Inducible 70-kDa Heat Shock Protein

doi: 10.3390/biom12060856

Figure Lengend Snippet: Phenyl arsine oxide (PAO) treatment significantly decreases HSPA1A’s PM localization after heat shock. ( A ) Representative images of HeLa cells expressing GFP-HSPA1A (top panel) and GFP-P4M-SidMx2 (second panel). Cells were either left untreated (DMSO; first row of each panel) or treated with 10nM PAO for 30 min (bottom row of each panel). Only heat-shocked cells are shown. For all cells, the PM was stained with WGA-FA555 and the nucleus with DAPI (second and third picture from the left of each panel). Scale bar = 10 μM. ( B ) Quantification of the corrected total cell fluorescence (CTCF) as a ratio between the total fluorescence at the PM and the rest of the cell at control conditions and after heat shock in the presence or absence of PAO. The experiment was repeated three times, and the total number of cells (shown as open circles) per condition are shown at the bottom of the graph; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; crosses represent sample means.

Article Snippet: The antibodies used were: the OmicsLinkTM Anti-GFP Tag Antibody Mouse Monoclonal IgG1 [(CGAB-GFP-0050, Genecopoeia, Rockville, MD, USA); 1:1000- detects a protein band of approximately 30 kDa (empty GFP), two bands of approximately 98 kDa (GFP-HSPA1A and GFP-EEA1), several bands around 52 kDa of the remaining biosensor constructs (Lact-C2-GFP, GFP-P4M-SidMx2, GFP-PLCδ-PH)]; the Anti-c-Myc Antibody (9E10, Genecopoeia, Rockville, MD, USA) [monoclonal IgG (Dilution 1:1000)- detects a protein of approximately 74 kDa (HSPA1A-myc)]; the anti-HSP70 Monoclonal Antibody [(mouse IgG) (Dilution 1:1000) clone #C92F3A-5 (Enzo Life Sciences, Farmingdale, NY, USA; detects a protein around 74 kDa; native and overexpressed HSPA1A)]; THETM beta actin antibody, GenScript [Piscataway, NJ, USA (mouse mAb; A00702) 1:1000- detects a protein band of approximately 42 kDa; cytosolic loading control]; and the Na+/K+ ATPase α (ATP1A1) antibody RabMAb ® [(EP1845Y); (2047-1), Epitomics, Burlingame, CA, USA; 1:1000- detects a protein band of approximately 112 kDa plasma membrane loading control].

Techniques: Expressing, Staining, Fluorescence, Control, Software

Journal: Biomolecules

Article Title: Phosphatidylinositol Monophosphates Regulate the Membrane Localization of HSPA1A, a Stress-Inducible 70-kDa Heat Shock Protein

doi: 10.3390/biom12060856

Figure Lengend Snippet: HSPA1A localization at the plasma membrane is significantly reduced by treatment with GSK-A1. Representative images of HeLa cells expressing ( A ) GFP-HSPA1A (top two rows), GFP-P4M-SidMx2 (bottom two rows), ( C ) GFP-PLCδ-PH (top two rows) and Lact-C2-GFP (bottom two rows). In all cases, the PM was stained with WGA-FA555 and the nucleus with DAPI. The PM localization of the proteins was documented at either control conditions (37 °C; left three columns) or heat-shocked conditions (1 h at 42 °C/8 h at 37 °C; right three columns). Negative control cells were treated with DMSO. Scale bar = 10 μM. ( B , D ) Quantification of the corrected total cell fluorescence (CTCF) as a ratio between the total GFP fluorescence of HSPA1A, P4M-SidMx2, PLCδ-PH, or Lact-C2 at the PM and the rest of the cell at control conditions and after heat shock in the presence of either DMSO (untreated) or GSK-A1. The experiment was repeated three times, and the total number of cells (shown as open circles) per condition are shown at the bottom of the graph. The center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; crosses represent sample means.

Article Snippet: The antibodies used were: the OmicsLinkTM Anti-GFP Tag Antibody Mouse Monoclonal IgG1 [(CGAB-GFP-0050, Genecopoeia, Rockville, MD, USA); 1:1000- detects a protein band of approximately 30 kDa (empty GFP), two bands of approximately 98 kDa (GFP-HSPA1A and GFP-EEA1), several bands around 52 kDa of the remaining biosensor constructs (Lact-C2-GFP, GFP-P4M-SidMx2, GFP-PLCδ-PH)]; the Anti-c-Myc Antibody (9E10, Genecopoeia, Rockville, MD, USA) [monoclonal IgG (Dilution 1:1000)- detects a protein of approximately 74 kDa (HSPA1A-myc)]; the anti-HSP70 Monoclonal Antibody [(mouse IgG) (Dilution 1:1000) clone #C92F3A-5 (Enzo Life Sciences, Farmingdale, NY, USA; detects a protein around 74 kDa; native and overexpressed HSPA1A)]; THETM beta actin antibody, GenScript [Piscataway, NJ, USA (mouse mAb; A00702) 1:1000- detects a protein band of approximately 42 kDa; cytosolic loading control]; and the Na+/K+ ATPase α (ATP1A1) antibody RabMAb ® [(EP1845Y); (2047-1), Epitomics, Burlingame, CA, USA; 1:1000- detects a protein band of approximately 112 kDa plasma membrane loading control].

Techniques: Clinical Proteomics, Membrane, Expressing, Staining, Control, Negative Control, Fluorescence, Software

Journal: Biomolecules

Article Title: Phosphatidylinositol Monophosphates Regulate the Membrane Localization of HSPA1A, a Stress-Inducible 70-kDa Heat Shock Protein

doi: 10.3390/biom12060856

Figure Lengend Snippet: HSPA1A localization at the plasma membrane is significantly reduced by treatment with wortmannin. ( A ) Representative images of HeLa cells expressing GFP-HSPA1A (first column from the left), Lact-C2-GFP (second column), GFP-P4M-SidMx2 (third column), GFP-PLCδ-PH (fourth column), and GFP-EEA1 (fifth column). In all cases, the PM was stained with WGA-FA555 and the nucleus with DAPI. The PM localization of the proteins was documented at either control conditions (37 °C; top panels) or heat-shocked conditions (1 h at 42 °C/8 h at 37 °C; bottom panels). Negative control cells were treated with DMSO. Scale bar = 10 μM. ( B ) Quantification of the corrected total cell fluorescence (CTCF) as a ratio between the total GFP fluorescence of HSPA1A, Lact-C2, P4M-SidMx2, or PLCδ-PH at the PM and the rest of the cell at control conditions and after heat shock in the presence of either DMSO (untreated) or wortmannin. Please note that the EEA1 cells were not quantified because they do not localize at the PM. The experiment was repeated three times, and the total number of cells (shown as open circles) per condition are shown at the bottom of the graph. The center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; crosses represent sample means.

Article Snippet: The antibodies used were: the OmicsLinkTM Anti-GFP Tag Antibody Mouse Monoclonal IgG1 [(CGAB-GFP-0050, Genecopoeia, Rockville, MD, USA); 1:1000- detects a protein band of approximately 30 kDa (empty GFP), two bands of approximately 98 kDa (GFP-HSPA1A and GFP-EEA1), several bands around 52 kDa of the remaining biosensor constructs (Lact-C2-GFP, GFP-P4M-SidMx2, GFP-PLCδ-PH)]; the Anti-c-Myc Antibody (9E10, Genecopoeia, Rockville, MD, USA) [monoclonal IgG (Dilution 1:1000)- detects a protein of approximately 74 kDa (HSPA1A-myc)]; the anti-HSP70 Monoclonal Antibody [(mouse IgG) (Dilution 1:1000) clone #C92F3A-5 (Enzo Life Sciences, Farmingdale, NY, USA; detects a protein around 74 kDa; native and overexpressed HSPA1A)]; THETM beta actin antibody, GenScript [Piscataway, NJ, USA (mouse mAb; A00702) 1:1000- detects a protein band of approximately 42 kDa; cytosolic loading control]; and the Na+/K+ ATPase α (ATP1A1) antibody RabMAb ® [(EP1845Y); (2047-1), Epitomics, Burlingame, CA, USA; 1:1000- detects a protein band of approximately 112 kDa plasma membrane loading control].

Techniques: Clinical Proteomics, Membrane, Expressing, Staining, Control, Negative Control, Fluorescence, Software

Journal: Biomolecules

Article Title: Phosphatidylinositol Monophosphates Regulate the Membrane Localization of HSPA1A, a Stress-Inducible 70-kDa Heat Shock Protein

doi: 10.3390/biom12060856

Figure Lengend Snippet: HSPA1A localization at the PM is decreased by the expression of Sac1 and WT enzymes. Representative images of HeLa cells expressing ( A ) GFP-HSPA1A, ( C ) GFP-P4M-SidMx2, and ( E ) GFP-PLCδ-PH with RFP-tagged PJ vectors and DAPI nucleus stain. The PM localization of HSPA1A was documented for all transfections at either control conditions (37 °C; left columns) or heat-shocked conditions (1 h at 42 °C/8 h at 37 °C; right columns). Negative control cells contain either the vehicle alone (Lyn11) or the inactive vector (Dead). Scale bar = 10 μM. ( B , D , F ) Quantification of the corrected total cell fluorescence (CTCF) as a ratio between the total GFP fluorescence of the protein of interest at the PM and the rest of the cell at control conditions and after heat shock with or without rapamycin treatment. The experiment was repeated three times, and the total number of cells (shown as open circles) per condition are shown at the bottom of the graph. The center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; crosses represent sample means.

Article Snippet: The antibodies used were: the OmicsLinkTM Anti-GFP Tag Antibody Mouse Monoclonal IgG1 [(CGAB-GFP-0050, Genecopoeia, Rockville, MD, USA); 1:1000- detects a protein band of approximately 30 kDa (empty GFP), two bands of approximately 98 kDa (GFP-HSPA1A and GFP-EEA1), several bands around 52 kDa of the remaining biosensor constructs (Lact-C2-GFP, GFP-P4M-SidMx2, GFP-PLCδ-PH)]; the Anti-c-Myc Antibody (9E10, Genecopoeia, Rockville, MD, USA) [monoclonal IgG (Dilution 1:1000)- detects a protein of approximately 74 kDa (HSPA1A-myc)]; the anti-HSP70 Monoclonal Antibody [(mouse IgG) (Dilution 1:1000) clone #C92F3A-5 (Enzo Life Sciences, Farmingdale, NY, USA; detects a protein around 74 kDa; native and overexpressed HSPA1A)]; THETM beta actin antibody, GenScript [Piscataway, NJ, USA (mouse mAb; A00702) 1:1000- detects a protein band of approximately 42 kDa; cytosolic loading control]; and the Na+/K+ ATPase α (ATP1A1) antibody RabMAb ® [(EP1845Y); (2047-1), Epitomics, Burlingame, CA, USA; 1:1000- detects a protein band of approximately 112 kDa plasma membrane loading control].

Techniques: Expressing, Staining, Transfection, Control, Negative Control, Plasmid Preparation, Fluorescence, Software