Journal: bioRxiv

Article Title: MRAP2 modifies the signaling and oligomerization state of the melanocortin-4 receptor

doi: 10.1101/2024.04.09.588099

Figure Lengend Snippet: (A) Representative images from TIRF microscopy of the basolateral membrane of HEK293AD cells expressing SNAP-tagged MC4R labeled using Alexa Fluor 647 (gray) and Nb35-eYFP (green). Images were taken pre-(top) and 4 min post-(bottom) stimulation with 1 µM NDP-α-MSH for cells expressing SNAP-MC4R alone or with MRAP2 (at a ratio of 1:4) and indicate recruitment of Nb35 (green intensity). (B) Kinetics of recruitment of Nb35 to the SNAP-MC4R–Gs complex, without (black; 5 transfections, 51 single cells) and with MRAP2 (1+4) (turquoise; 5 transfections, 55 cells). Shown are the normalized fluorescence intensities recorded at the membrane in the eYFP channel; the shaded area represents SEM. (C) Mean concentration-response curves of Rluc8-miniGs recruitment to the plasma membrane localization sensor rGFP-CAAX upon MC4R stimulation with α-MSH for 45 minutes, in the presence or absence of MRAP2 or RAMP3, at the indicated ratio with respect to MC4R, in transiently transfected HEK293-SL cells. Normalized data are expressed as mean ± SEM of three independent experiments. (D) EC50 values from the miniGs recruitment BRET experiments. Data are expressed as mean ± SEM of three independent experiments. Statistical analysis was performed using ordinary one-way ANOVA with Dunnett’s multiple comparisons post-hoc test (*** = p < 0.001).

Article Snippet: Prior to microscopy imaging experiments, coverslips with cells expressing SNAP-MC4R and MRAP2 were labeled with 1 μM SNAP-Surface Alexa Fluor 647 dye in complete DMEM (Pan Biotech) for 25 min and kept in the incubator at 37 °C and 5 % CO 2 .

Techniques: Microscopy, Membrane, Expressing, Labeling, Transfection, Fluorescence, Concentration Assay

Journal: Oncotarget

Article Title: Isolation and detection of circulating tumour cells from metastatic melanoma patients using a slanted spiral microfluidic device

doi: 10.18632/oncotarget.18641

Figure Lengend Snippet: (A) Optimisation of immunostaining protocol in WBC samples spiked with A2058 cells, cytospun onto glass slides, fixed, permeabilised and immunostained with a combination of cell surface, MCSP (Alexa Fluor® 647, purple), and intracellular -MEL- (gp100, S100 and MLANA; Alexa Fluor® 488, green) markers. DAPI was used for nuclei staining and CD45 (PE, red) as a leukocytic marker. Differential expression of MCSP and MEL markers was observed in A2058 cells. (B) Representative melanoma CTCs detected by this immunofluorescence staining in metastatic melanoma patient blood processed twice through the slanted spiral microfluidic device. Melanoma CTCs had an average cell size of 16 μm (range: 13 – 21 μm). MCSP staining was not detected in any of the isolated melanoma CTCs from patient samples. Scale bar represents size of the cell in μm.

Article Snippet: Cells were then stained in a 1 hour incubation with blocking buffer and a cocktail of the following antibodies: rabbit anti-gp100 (clone EPR4864, Abcam), rabbit anti-MLANA/MART1 (clone EP1422Y, Abcam), rabbit anti-S100 (clone EP1576Y, Abcam), mouse anti-MCSP conjugated to Alexa Fluor® 647 dye (clone 9.2.27, BD Pharmingen ™ ) and mouse anti-CD45 conjugated to Phycoerythrin (PE) (clone HI30, BD Pharmingen ™ ).

Techniques: Immunostaining, Staining, Marker, Expressing, Immunofluorescence, Isolation