flag-mal2  (Genechem)

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: (A) Immunofluorescence staining for HER2 and cholera toxin B in MCF10A and SKBR3 cells. Scale bars represent 10 μm. (B) Lipid raft areas on the cell surface, with quantification in the bar graph on the right. (C) Flotillin1 (FLOT1), MAL, and MAL2 mRNA expression in different breast cancer cell lines as assessed by quantitative PCR (n = 3). (D) RNA-seq analysis of FLOT1 and MAL2 expression in normal breast tissue (n = 112) and HER2-positive breast tumors (n = 160) represented in The Cancer Genome Atlas database. (E) Uniform Manifold Approximation and Projection (UMAP) plots of breast cancer single-cell RNA-seq data (GEO: GSE75688, left) and co-expression pattern of HER2, FLOT1, MAL, and MAL2 in cells from cluster 2. (F) Distribution of MAL, FLOT1, MAL2, and HER2 expression level for each cell in cluster 2. (G) MAL2 ATAC-seq peak clusters in SKBR3, MCF10A, MCF7, and MDA-MB-231 cell lines. In the bar graphs, the bars represent the mean ± SEM. **p <0.01, ***p <0.001, ****p <0.0001. These results are representative of three independent experiments.

Article Snippet: Constructs encoding flag-flotillin1 (RC200231) and flag-MAL2 (RC203862) are commercially available from OriGene (Rockville, MD).

Techniques: Immunofluorescence, Staining, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: (A) Immunofluorescence staining for HER2 and MAL2 (top row) and MAL2 and cholera toxin B (lipid rafts) (bottom row) in SKBR3 cells. (B) Immunofluorescence staining for FLAG-tagged MAL2 or endogenous MAL2 with HER2 and actin in control and MβCD (5 mM)-treated SKBR3 cells. Scale bars represent 10 μm. (C) Proximity ligation assay (PLA) experiment for HER2 and MAL2 in control and MβCD (5 mM)-treated SKBR3 cells also stained for actin (phalloidin). Scale bars represent 10 μm. (D) MAL2 mRNA expression in control and MAL2 knockdown SKBR3 and BT474 cells as assessed by quantitative RT-PCR (n = 3). (E) Western blot analysis of HER2, phospho-HER2, EGFR, and phospho-EGFR in control and MAL2 knockdown SKBR3 and BT474 cells. (F) BrdU incorporation in MAL2KD cells relative to control SKBR3 and BT474 cells. (G) PLA experiment for HER2 and MAL2 in control and MAL2LD SKBR3 cells. Scale bars represent 10 μm. (H) Transmission electron microscopy images in control and MAL2 knockdown SKBR3 cells. (I) Immunofluorescence staining HER2 and cholera toxin B in control (top), MAL2KD-treated (middle), and MβCD-treated (5 mM) SKBR3 cells. Scale bars represent 10 μm. (J) Immunofluorescence staining for FLAG-tagged FLOT1, HER2, and phalloidin in control, MAL2KD-treated, and MβCD (5 mM)-treated SKBR3 cells. Scale bars represent 10 μm. (K) Immunofluorescence staining for HER2 and EGFR in control and MAL2KD SKBR3 cells. Scale bars represent 10 μm. (L) PLA experiment for HER2 and EGFR in control and MAL2KD SKBR3 cells also stained for actin (phalloidin). Scale bars represent 10μm. (M) Quantitation of PLA experiment by measuring fluorescent intensity of amplified PLA reactions and phalloidin. Bar graphs represent the mean ± SEM. **p <0.01, ***p <0.001, ****p <0.0001. These results are representative of three independent experiments.

Article Snippet: Constructs encoding flag-flotillin1 (RC200231) and flag-MAL2 (RC203862) are commercially available from OriGene (Rockville, MD).

Techniques: Immunofluorescence, Staining, Proximity Ligation Assay, Expressing, Quantitative RT-PCR, Western Blot, BrdU Incorporation Assay, Transmission Assay, Electron Microscopy, Quantitation Assay, Amplification

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: (A) SIM imaging of fluorescent staining for HER2, Ezrin, and phalloidin in control SKBR3 cells. Images represent different combinations of staining as noted on the figures. Bottom left: a 3D reconstruction for all three molecules. Scale bars represent 10 μm. (B) SIM imaging of fluorescent staining for HER2, HA-tagged NHERF1, and phalloidin in control SKBR3 cells. Bottom left: a 3D reconstruction for all three molecules. Scale bars represent 10 μm. (C) SIM imaging of fluorescent staining for HER2, Ezrin, and phalloidin in MAL2 knockdown SKBR3 cells. Scale bars represent 10 μm. (D) SIM imaging of fluorescent staining for HER2, HA-tagged NHERF1, and phalloidin in MAL2 knockdown SKBR3 cells. Scale bars represent 10 μm. (E) PLA for HER2 with Ezrin (left panels) or NHERF1 (right panels) in control (top row), MAL2 knockdown-treated (middle row) and MβCD-treated (bottom row) SKBR3 cells. Columns labeled “Middle” represent an optical section through the mid-portion of the cells. Columns labeled “Top“ represent an optical section taken near the apical surface of the cell. Columns labeled “w/Phalloidin” represent co-registration of the PLA signal and immunofluorescence for actin (phalloidin). Scale bars represent 10 μm (for all images). (F) Percentage of cells with positive PLA reactions located within membrane protrusions. (G) Quantitation of PLA fluorescent intensity within membrane protrusions. Bar graphs represent the mean ± SEM. ****p <0.0001. These results are representative of three independent experiments.

Article Snippet: Constructs encoding flag-flotillin1 (RC200231) and flag-MAL2 (RC203862) are commercially available from OriGene (Rockville, MD).

Techniques: Imaging, Staining, Labeling, Immunofluorescence, Quantitation Assay

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: (A) Co-immunofluorescence staining for Ezrin (top) or HER2 (bottom) with phalloidin in PH-PLCδ-GFP-expressing control SKBR3 cells. Scale bars represent 10 μm. (B) Immunofluorescence staining for Ezrin (top) or HER2 (bottom) with phalloidin in PH-PLCδ-GFP-expressing MAL2KD_SKBR3 cells. (C) Immunofluorescence staining for Ezrin (top) or HER2 (bottom) with phalloidin in PH-PLCδ-GFP-expressing SKBR3 cells treated with MβCD. (D) Immunofluorescence staining for Ezrin with phalloidin in PH-PLCδ-GFP-expressing SKBR3 cells treated with wortmannin (0, 5, and 10 μM). (E) Immunofluorescence staining for HER2 with phalloidin in PH-PLCδ-GFP-expressing SKBR3 cells treated with wortmannin (0, 5, and 10 μM). (F) PLA results for HER2 and Ezrin in control and wortmannin-treated SKBR3 cells. Scale bars represent 10 μm. (G) Western blot analysis of AKT and phospho-AKT in control and MAL2 knockdown SKBR3 cells (left) and in control and MβCD-treated SKBR3 cells (right). (H–J) SIM imaging showing HER2, pAKT, and phalloidin immunofluorescence in control (H), MAL2KD (I)-treated, and MβCD-treated (J) SKBR3 cells. Scale bars represent 10 μm. (K) Immunofluorescence staining for FOXO1 in control, MAL2KD-treated, and MβCD-treated SKBR3 cells. (L) XTT cell viability assay in control, MAL2KD-treated, and MβCD-treated SKBR3 and BT474 cells. Bar graphs represent the mean ± SEM. ****p <0.0001. These results are representative of three independent experiments.

Article Snippet: Constructs encoding flag-flotillin1 (RC200231) and flag-MAL2 (RC203862) are commercially available from OriGene (Rockville, MD).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Imaging, Viability Assay

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: (A) Immunofluorescence staining for cholera toxin B (lipid rafts) in control and trastuzumab-resistant SKBR3 cells. Scale bars represent 10 μm. (B) Immunofluorescence staining for HER2 and MAL2 in control and trastuzumab-resistant SKBR3 cells. Scale bars represent 10 μm. (C) PLA for HER2 and MAL2 in control and trastuzumab-resistant SKBR3 cells also stained for phalloidin. Scale bars represent 10 μm. (D–F) Quantitative results from immunoprecipitation coupled with data-independent acquisition mass spectrometry (DIA-MS) in control and trastuzumab-resistant SKBR3 cells. (D) The DIA-MS Intensity (log 2 ) of HER2 and MAL2 proteins from control and trastuzumab-resistant SKBR3 cells. (E) The DIA-MS Intensity (log 2 ) for all the peptide precursor signals of HER2 in control and resistant cells. (F) The DIA-MS peak groups visualized for quantifying HER2 (VLGSGAFGTVYK) and MAL2 (VTLPAGPDILR). Peaks above and below the middle line denote the MS2 and MS1 ion traces in DIA-MS. (G) MAL2, Ezrin, and NHERF1 mRNA expression in control and trastuzumab-resistant SKBR3 cells as assessed by quantitative RT-PCR (n = 3). (H) PLA for HER2 with Ezrin (left), NHERF1 (middle), and PMCA2 (right) in control and trastuzumab-resistant SKBR3 cells also stained for phalloidin. Boxed portions are amplified at right with co-registration of PLA signal and immunofluorescence for actin (phalloidin). (I) Quantitation of PLA experiment for HER2 in combination with MAL2, Ezrin, NHERF1, or PMCA2 represented as the fluorescent intensity of amplified PLA signals associated with membrane protrusions. (J) Coimmunoprecipitation for HER2 and HSP90 in control and trastuzumab-resistant SKBR3 cells. (K) PLA for HER2 and HSP90 in control and trastuzumab-resistant SKBR3 cells also stained for phalloidin. Scale bars represent 10 μm. (L) XTT cell viability assay in control, MAL2KD-treated, and MβCD-treated trastuzumab-resistant SKBR3 and BT474 cells. (M) Immunofluorescence staining for FOXO1 in control, MAL2KD-treated, and MβCD-treated trastuzumab-resistant SKBR3 cells. (N) Immunofluorescence staining for HER2 and pAKT in control, MAL2KD-treated, and MβCD-treated trastuzumab-resistant SKBR3 cells. Scale bars represent 10 μm. (O) Diagram representing the structure of MAL2- and lipid raft-enriched membrane protrusions containing multi-protein HER2 signaling complexes. These results are representative of three independent experiments.

Article Snippet: Constructs encoding flag-flotillin1 (RC200231) and flag-MAL2 (RC203862) are commercially available from OriGene (Rockville, MD).

Techniques: Immunofluorescence, Staining, Immunoprecipitation, Mass Spectrometry, Expressing, Quantitative RT-PCR, Amplification, Quantitation Assay, Viability Assay

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Constructs encoding flag-flotillin1 (RC200231) and flag-MAL2 (RC203862) are commercially available from OriGene (Rockville, MD).

Techniques: Recombinant, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Sequencing, Microarray, Software, Imaging, Light Microscopy