Journal: Signal Transduction and Targeted Therapy

Article Title: Soluble CD4 effectively prevents excessive TLR activation of resident macrophages in the onset of sepsis

doi: 10.1038/s41392-023-01438-z

Figure Lengend Snippet: sCD4 disrupted MHCII/TLR4 rafts and reduced LPS/TLR4 inflammatory membrane confinement. Duolink assays to quantify protein-protein interactions of ( a ) the indicated pairs (red), or ( b ) between STING and SHP2 (green) that combined with immunofluorescent staining of MHC II (red) in BMDM. Tripartite colocalization indicated in yellow. The nuclei counter-stained with DAPI. Pearson’s coefficients indicated the degree of colocalization. Bar = 5 μm. c TNF and IL-6 in supernatants 30 min after peritoneal macrophages treated with LPS or LPS plus sCD4, in the presence of indicated endocytosis inhibitors. The average of two independent repeats. Three replicate wells were used for each condition where the indicated inhibitor was added. d Duolink spots of MHC II-SHP2 interactions and ( e ) flow cytometric analyses of cell surface MHC II. Bar = 5 μm. Macrophages (5 x 10 6 ) were treated with LPS/sCD4 for 1 h, and ( f ) endosomes were isolated for immunoblot analysis of the indicated proteins (asterisk), or ( g ) organelle numbers per cell were quantified after immunofluorescence staining of EEA1 (early endosomes), LAMP1 (lysosomes), RAB4 (recycling endosome) and RCAS1 (Golgi). h Lysosomes size was quantified using LysoTracker after RAW264.7 cells were transfected with GFP-tagged MHC II Aβ or MHC II AβΔCT. Several view fields were randomly selected and images were acquired every 10 s for 20 min of LPS or LPS plus sCD4 treatment. Mean ± SD are shown; n = 3–4 mice used where indicated; Statistics (ns, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001;): Unpaired t test

Article Snippet: Antibodies against indicated proteins used in this study were: CD4 (GK1.5 and RM4-5) from eBioscience, mouse IgG2a isotype control antibody (401501) and MHC class II (107610) from BioLegend; TRIF (ab13810), β-actin, Goat anti-Rat Alexa Fluor 647(A-21247), Donkey anti-Mouse IgG Alexa Fluor 488(A-21202) and Goat anti-Rabbit Alexa Fluor 555(A-21429) from Thermo Fisher; TRAF6 (sc-8409) from Santa Cruz; MyD88 (D80F5), IRF3 (D83B9), Syk (D3Z1E), Btk (D3H5), IRAK1 (D51G7), Erk (9102), IgG (7074, 7076), Jnk (56G8), p38 (9212), SHP-2 (D50F2), phospho-Erk at Thr202-Tyr204 (E10), phospho-Jnk at Thr183-Tyr185 (G9), phospho-SHP-2 at Tyr580 (5431), phospho-p38 at Thr180-Tyr182 (9211), phospho-IRF3 at Ser396 (4D4G), phospho-IκBα at Ser32-Ser36 (5A5), phospho-Btk at Tyr223(D9T6H), phospho-Syk at (Tyr352), phospho-Tyrosine antibody (p-Tyr-100), EEAL (C45B10), LAPM1 (D2D11), RAB4 (2167T) and RCAS1(D2B6N) from Cell Signaling Technology; RAB5(AR038) from Beyotime.

Techniques: Membrane, Protein-Protein interactions, Staining, Isolation, Western Blot, Immunofluorescence, Transfection

Journal: Developmental cell

Article Title: Membrane flow drives an adhesion-independent amoeboid cell migration mode

doi: 10.1016/j.devcel.2018.05.029

Figure Lengend Snippet: Data and Software Availability

Article Snippet: Method Details DNA Constructs The following constructs were obtained from Addgene: mVenus-myosinIIA (#56389, Michael Davidson), GFP-rGBD (#26732, William Bement), mCherry-ezrin (#55043, Michael Davidson), mCherry-moesin (#55103, Michael Davidson), Venus-iLID-CaaX (#60411, Brian Kuhlman), mRFP-FKBP12-5ptpase (#67516, Tamas Balla), mRFP-FKBP12 (#67514, Tamas Balla), EYFP-Clathrin-19 (#56584, Michael Davidson), mCherry-Lifeact-7 (#54491, Michael Davidson), mApple-caveolin (#54872, Michael Davidson), pUASP-YFP-Rab4 (#37690, Matthew Scott), pUASP-YFP-Rab4DN (#37691, Matthew Scott).

Techniques: Software, Recombinant, Sequencing, RNA Sequencing Assay

Journal: Frontiers in Immunology

Article Title: Chlamydia trachomatis Infection Impairs MHC-I Intracellular Trafficking and Antigen Cross-Presentation by Dendritic Cells

doi: 10.3389/fimmu.2021.662096

Figure Lengend Snippet: C. trachomatis recruits Rab14, Rab4, Rab22a, and Rab11a to the inclusion in JAWS-II DCs. JAWS-II DCs were infected with GFP- C. trachomatis L2 at MOI 100 for 24 h and analyzed by confocal microscopy. The intracellular distribution of (A, B) Rab14, (E, F) Rab4, (I, J) Rab22a and (M, N) Rab11a was evaluated in non-infected and infected cells. Endogenous Rab proteins were detected by indirect immunofluorescence using primary antibodies followed by its corresponding Cy3-conjugated secondary antibodies. Insets show a chlamydial inclusion magnification for each marker. DAPI was used to stain DNA. Bars represent 10 um. Images are representative of three independent experiments and more than 20 images were analyzed for each Rab. (C, G, K, O) A representative chlamydial inclusion was transversely crossed with eight diameter lines to obtain an intensity histogram. Line graphs show the MFI from GFP and the corresponding Rab protein with the SD for each point. (D, H, L, P) Chlamydial inclusion was transversely crossed with a diameter line to obtain an intensity histogram for each Rab protein; and five points of each line corresponding to the cytoplasm (P1), inclusion membrane (P2), inside the inclusion (P3), inclusion membrane (P4), and cytoplasm (P5) were selected. Twenty cells were analyzed for each Rab protein. One-way ANOVA with Dunnett’s multiple comparison post-test were performed. ### P < 0.001, #### P < 0.0001, and ****P < 0.0001.

Article Snippet: The following primaries antibodies were used: rabbit anti-CT529 (gently provided by Agathe Subtil, Pasteur Institute, Paris, France), rabbit polyclonal anti-Rab14, Rab4 and Rab11a (Aviva Systems Biology), mouse anti-CD63 (Invitrogen), goat polyclonal anti-EEA1 (Santa Cruz), purified mouse anti-Lamp1, purified mouse anti-H-2K b and FITC-coupled mouse anti-H-2K b (BD Biosciences), mouse monoclonal anti-TfR H68.4 (Invitrogen), mouse monoclonal anti-Rab22a (Santa Cruz), rabbit anti-clathrin (Abcam), rabbit anti-OVA (Sigma-Aldrich), and rabbit anti-MOMP (generously provided by Ted Hackstadt, National Institutes of Health, USA).

Techniques: Infection, Confocal Microscopy, Immunofluorescence, Marker, Staining, Membrane, Comparison

Journal: Cell reports

Article Title: Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina.

doi: 10.1016/j.celrep.2024.114005

Figure Lengend Snippet: Figure 3. Co-localization of MEGF10 and FAT3 with ChAT is disrupted in PtencKO retinas (A–C) Colabeling of P14 wild-type retinas with CD63 (blue), ChAT (green), FAT3 (red, A), or MEGF10 (red, B). Arrows point to CD63 puncta that are positive for FAT3 or MEGF10. Also shown is a schematic of CD63+ endosomal compartments (C). (D–F) Colabeling of P14 wild-type retinas with RAB4 (blue), ChAT (green), FAT3 (red, D), and MEGF10 (red, E). Arrows point to RAB4 puncta that are positive for FAT3 or MEGF10. Also shown is a schematic of RAB4+ endosomal compartments (F). (G–I) Colabeling of P14 wild-type retinas with RAB5 (blue), ChAT (green), FAT3 (red, G) or MEGF10 (red, H). Arrows point to RAB5 puncta that are positive for FAT3 or MEGF10. Also shown is a schematic of RAB5+ endosomal compartments (I). (J–O) Colabeling of P14 wild-type (J and M) and PtencKO (K and N) retinas with ChAT (green) and MEGF10 (red, J and K) or FAT3 (red, M and N). Blue is DAPI counterstain. Also shown is quantification of MEGF10+ (L) and FAT3+ (O) puncta in ChAT+ cell somata in wild-type and PtencKO retinas. Plots show means ± SEM. N = 3 biological replicates/genotype, all with 3 technical replicates, with 50 total cells analyzed for wild-type and 48 cells for PtencKO. The p values were calculated with an unpaired t test. Scale bars: 10 mm (A–H), 40 mm (J–N in the first), and 10 mm in the remaining. See also Figure S4.

Article Snippet: The cells were also immunostained with RAB4 antibody (Novus Biologicals, NBP2-37485) and a secondary antibody.

Techniques:

Journal: Cell reports

Article Title: Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina.

doi: 10.1016/j.celrep.2024.114005

Figure Lengend Snippet: Figure 4. CAM endocytic recycling is accelerated in PtencKO retinas (A) Summary of the CAM endocytosis assay, showing immunolabeling and quantification of VC.1.1+ and Pax6+ MACS-enriched amacrine cells. Plots show means ± SEM. N = 3 biological replicates per sample from one experiment. (B) Western blot of pAktSer473 in P7 retinal explants treated for 1 day with 1 mm or 10 mm bpV(pic). Densitometry is normalized to actin beta (ACTB). The plot shows means ± SEM. N = 3 biological replicates per sample from one experiment. p values were calculated with an unpaired t test. (C–E) Immunolabeling of RAB4 with secondary antibodies to biotin-conjugated DSCAM (C), FAT3 (D), or MEGF10 (E) in MACS-enriched amacrine cells treated with DMSO or bpV(pic) for 1 h, 3 h, and 12 h. (F) Quantification of the overlapping relative fluorescence signals of RAB4 with DSCAM, MEGF10, and FAT3. Plots show means ± SEM. 27–37 individual cells collected from 3 biological replicates were analyzed per time point and condition in 3 independent experiments. The p values were calculated with an unpaired t test in pairwise comparisons at each time point. (G) Schematic highlighting how Pten affects endocytic trafficking. Scale bars: 20 mm (C–E). See Figure S5.

Article Snippet: The cells were also immunostained with RAB4 antibody (Novus Biologicals, NBP2-37485) and a secondary antibody.

Techniques: Endocytosis Assay, Immunolabeling, Western Blot