Journal: bioRxiv

Article Title: Flotillin-2 regulates EGFR activation, degradation, and cancer growth

doi: 10.1101/2022.03.11.483779

Figure Lengend Snippet: (A) Control (Vector) and FLAG-FLOT2 overexpressing (WT) clones were created in HeLa cells and whole-cell lysates (WCL, upper panel) were analyzed on western blot with the indicated antibodies. EGFR was immunoprecipitated (IP EGFR, lower panel) and analyzed for phosphorylation (pY 4G10 AB), ubiquitination (Ub), interaction with FLOT2 and EGFR levels. (B) The graph shows the average (± SEM) EGFR phosphorylation at Y1068 for six experiments calculated as a ratio of the intensity of pEGFR to EGFR bands on WCL as in A. (C) The graph shows the average (± SEM) EGFR ubiquitination for five experiments calculated as a ratio of the intensity of Ub to EGFR bands on IP EGFR as in A. (D) Vector, FLAG-tagged WT FLOT2 and FLOT2 mutants (G2A and Y163F) were overexpressed in HEK293T cells for 48 hours and the cells were stimulated with 100 ng/mL EGF as indicated. The lysates (WCL, upper panel) as well as EGFR IP (lower panel) were analyzed on western blot with the indicated antibodies. (E) The effect of FLOT2 overexpression on EGFR phosphorylation was assessed by normalizing the intensities of pEGFR bands to corresponding EGFR bands of WCL as in D. The graph shows the average (± SEM) of three independent experiments. In all panels, asterisk (*) denotes p < 0.05, (**) indicates p < 0.01 as compared to the corresponding time points of Vector (except E) using Student’s t -test. MW in kDa is shown to the left of the western blot panels.

Article Snippet: Plasmids for overexpression in HEK293T cells were purchased from OriGene (Rockville, MD): pCMV6 (PS10001), FLOT1 (RC200231), FLOT2 (RC220884).

Techniques: Control, Plasmid Preparation, Clone Assay, Western Blot, Immunoprecipitation, Phospho-proteomics, Ubiquitin Proteomics, Over Expression

Journal: Frontiers in cell and developmental biology

Article Title: Differential Effects of APOE Genotype on MicroRNA Cargo of Cerebrospinal Fluid Extracellular Vesicles in Females With Alzheimer's Disease Compared to Males.

doi: 10.3389/fcell.2022.864022

Figure Lengend Snippet: FIGURE 3 | SEC qEV single 35 nm columns are optimal for separating CSF EVs. Pools of SEC fractions (Fxs): 1–5 (column void volume), 6–9, 10–13, and 14–17 were generated using either the qEV Single 35 nm or 70 nm columns. (A) Equal concentration loading of protein lysate (0.1 µg) from SEC pools immunoblotted for flotillin and CD81. (B) Equal volume loading of protein lysate (37 µL) from SEC pools immunoblotted for APOA1 and albumin. (C) Size and concentration histograms of Fxs 6–9 generated using either the qEV Single 35 nm (white) or 70 nm (black) columns acquired by tunable resistive pulse sensing (TRPS).

Article Snippet: The following antibodies were used for immunoblotting: albumin 1:1,000 (#4929, Cell Signaling Technology, Danvers, MA), APOA1 (12C8) 1:200 (sc-080551, Santa Cruz Biotechnology, Dallas, TX), APOE 1:2000 (50A-G1A, Academy Bio-medical Company, Inc., Houston, TX), AnnV 1: 5,000 (GTX103250, GeneTex, Irvine, CA), CD9 (C-4) 1:200 (sc13118, Santa Cruz Biotechnology), CD11b 1:1,000 (ab133357, Abcam, Cambridge, United Kingdom), CD63 1:1,000 (ab134045, Abcam), CD81 (B-11) 1:100 (sc-166029, Santa Cruz Biotechnology), flotillin 1:10,000 (ab133497, Abcam), GLAST 1:500 (NB100-1869, Novus Biologicals, Littleton, CO), NCAM1 1:125 (NBP2-38452, Novus Biologicals), SYP 1:1,000 (#36406, Cell Signaling Technology), TMEM119 (#66948-1-Ig, Proteintech, Rosemont, IL), and TSG101 1:1,000 (ab125011, Abcam).

Techniques: Generated, Concentration Assay, Tunable Resistive Pulse Sensing

Journal: Frontiers in cell and developmental biology

Article Title: Differential Effects of APOE Genotype on MicroRNA Cargo of Cerebrospinal Fluid Extracellular Vesicles in Females With Alzheimer's Disease Compared to Males.

doi: 10.3389/fcell.2022.864022

Figure Lengend Snippet: FIGURE 5 | CSF EVs isolated by SEC are enriched for exosome and MV markers. (A) Equal volume loading of protein lysate (37 µL) from pools of SEC Fractions (Fxs): 1–5 (column void volume), 6–9, 10–13, and 14–17 stained for total protein, and immunoblotted for albumin, APOA1, and APOE. (B) Equal concentration loading of protein lysate (0.1 µg) from pools of Fxs 1–5, 6–9, 10–13, and 14–17 immunoblotted for CD9, CD63, CD81, flotillin, TSG101, AnnV, SYP, NCAM-1, GLAST, CD11b, and TMEM119. (C) Equal concentration loading of (1 µg) from pools of Fxs 1–5, 6–9, 10–13, and 14–17 immunoblotted for SYP and CD11b. Postmortem human cerebral cortex protein lysate (0.1 µg and 1 µg) was run as a positive control for each gel ((A-C): Brain).

Article Snippet: The following antibodies were used for immunoblotting: albumin 1:1,000 (#4929, Cell Signaling Technology, Danvers, MA), APOA1 (12C8) 1:200 (sc-080551, Santa Cruz Biotechnology, Dallas, TX), APOE 1:2000 (50A-G1A, Academy Bio-medical Company, Inc., Houston, TX), AnnV 1: 5,000 (GTX103250, GeneTex, Irvine, CA), CD9 (C-4) 1:200 (sc13118, Santa Cruz Biotechnology), CD11b 1:1,000 (ab133357, Abcam, Cambridge, United Kingdom), CD63 1:1,000 (ab134045, Abcam), CD81 (B-11) 1:100 (sc-166029, Santa Cruz Biotechnology), flotillin 1:10,000 (ab133497, Abcam), GLAST 1:500 (NB100-1869, Novus Biologicals, Littleton, CO), NCAM1 1:125 (NBP2-38452, Novus Biologicals), SYP 1:1,000 (#36406, Cell Signaling Technology), TMEM119 (#66948-1-Ig, Proteintech, Rosemont, IL), and TSG101 1:1,000 (ab125011, Abcam).

Techniques: Isolation, Staining, Concentration Assay, Positive Control