Journal: eLife

Article Title: A hierarchical pathway for assembly of the distal appendages that organize primary cilia

doi: 10.7554/eLife.85999

Figure Lengend Snippet: ( A ) and ( B ) 3D single-molecule data from showing FOP data from the two channels separately. The manually isolated localizations of CF568-labeled FOP (blue) and Alexa Fluor 647 (AF647)-labeled FOP (orange), shown for top and side views, along with CF568-labeled MYO5A (green) and AF647-labeled RAB34 (magenta) for the two samples. Orientations in the microscope 3D space are indicated by the inset axes. The opacities used for visualization in Vutara SRX are as follows: Sample 1, RAB34: 0.03, MYO5A: 0.08, FOP-CF568: 0.05, FOP-AF647: 0.05; sample 2, RAB34: 0.02, MYO5A: 0.09, FOP-CF568: 0.05, FOP-AF647: 0.05. ( C–F ) Example of reconstructions of the FOP ring structures in the two color channels after channel transformation and cross-correlation of FOP that is labeled in both channels. ( C–D ) Example of the AF647-labeled (magenta) and CF568-labeled (green) FOP structures at the mother and daughter centrioles used for cross-correlation. ( E–F ) The FOP structures from the two channels after channel transformation and data cross-correlation. The opacities for the visualization are set in Vutara SRX to 0.07 for AF647 FOP and 0.02 for CF568 FOP.

Article Snippet: In the experiments shown in , the cells were incubated with rabbit anti-RAB34 (27435–1-AP, Proteintech, 1:500), mouse IgG2a anti-RAB34 (sc-365986, Santa Cruz, 1:250), and mouse IgG2b anti-FOP (H00011116-M01, Abnova, 1:1000) diluted in 1% BSA in PBS at 4 °C overnight, washed three times in 0.1% Triton-X 100 in PBS, and then incubated with donkey anti-rabbit Alexa Fluor 647 (ab150067, Abcam, 1:1000), goat anti-mouse IgG2b Alexa Fluor 647 (A-21242, Invitrogen), goat anti-mouse IgG2a CF568 (20258, Biotium), and goat anti-mouse IgG2b CF568 (20268, Biotium) diluted in 1% BSA in PBS for 1 hr shielded from light.

Techniques: Isolation, Labeling, Microscopy, Transformation Assay

Journal: eLife

Article Title: A hierarchical pathway for assembly of the distal appendages that organize primary cilia

doi: 10.7554/eLife.85999

Figure Lengend Snippet: ( A ) Super-resolution reconstructions from the localizations of CF568-labeled RAB34 and Alexa Fluor 647 (AF647)-labeled RAB34. The double-sided arrows specify the axes of the histograms shown in ( B ). ( B ) Offsets between the center of masses of CF568-labeled RAB34 and AF647-labeled RAB34 were found by fitting normalized histograms along each axis to Gaussian functions and calculating the difference between the two peaks. This was done in the x, y, and z directions before finding the total offset in 3D space. The offset between the localizations was found to be 20 nm in this sample, and there were 1705 localizations in the red channel and 1228 localizations in the green channel for RAB34. ( C ) Super-resolution reconstruction of RAB34 and FOP in both channels shown for top view (top) and side view (bottom) after channel registration. Visualizations were rendered in Vutara SRX as 3D Gaussians with 20 nm diameter. The opacities used for visualization are as follows: ( A ) RAB34-Alexa647: 0.06, RAB34-CF568: 0.06; ( C ) RAB34-AF647: 0.1, RAB34-CF568: 0.1, FOP-AF647: 0.05, FOP-CF568: 0.05.

Article Snippet: In the experiments shown in , the cells were incubated with rabbit anti-RAB34 (27435–1-AP, Proteintech, 1:500), mouse IgG2a anti-RAB34 (sc-365986, Santa Cruz, 1:250), and mouse IgG2b anti-FOP (H00011116-M01, Abnova, 1:1000) diluted in 1% BSA in PBS at 4 °C overnight, washed three times in 0.1% Triton-X 100 in PBS, and then incubated with donkey anti-rabbit Alexa Fluor 647 (ab150067, Abcam, 1:1000), goat anti-mouse IgG2b Alexa Fluor 647 (A-21242, Invitrogen), goat anti-mouse IgG2a CF568 (20258, Biotium), and goat anti-mouse IgG2b CF568 (20268, Biotium) diluted in 1% BSA in PBS for 1 hr shielded from light.

Techniques: Labeling

Journal: eLife

Article Title: A hierarchical pathway for assembly of the distal appendages that organize primary cilia

doi: 10.7554/eLife.85999

Figure Lengend Snippet: Single fluorophores of CF568 and AF647 are excited by 560 nm and 642 nm lasers, respectively, using widefield epi-illumination. A 100 x objective lens serves for both illumination and collection of emitted light. A 4 f optical relay system is used to image the emitted light, which is split by a dichroic mirror into two emission paths. Transmissive phase masks in the Fourier plane of both paths modulate the emission light, thereby changing the shape of the point spread function (PSF) to that of the double helix PSF, which encodes the 3D position of the emitter. The two paths which are split in the 4 f system are imaged onto separate regions of an EMCCD camera. FOP is labeled with both dyes and is imaged in both channels, whereas MYO5A and RAB34 are labeled and imaged in channels 1 and 2, respectively, as indicated above. The inset shows the shape of the double helix PSF in both channels at different axial ( z ) positions. Scale bar is 1 µm. The schematic is not drawn to scale.

Article Snippet: In the experiments shown in , the cells were incubated with rabbit anti-RAB34 (27435–1-AP, Proteintech, 1:500), mouse IgG2a anti-RAB34 (sc-365986, Santa Cruz, 1:250), and mouse IgG2b anti-FOP (H00011116-M01, Abnova, 1:1000) diluted in 1% BSA in PBS at 4 °C overnight, washed three times in 0.1% Triton-X 100 in PBS, and then incubated with donkey anti-rabbit Alexa Fluor 647 (ab150067, Abcam, 1:1000), goat anti-mouse IgG2b Alexa Fluor 647 (A-21242, Invitrogen), goat anti-mouse IgG2a CF568 (20258, Biotium), and goat anti-mouse IgG2b CF568 (20268, Biotium) diluted in 1% BSA in PBS for 1 hr shielded from light.

Techniques: Labeling

Journal: Stem cells and development

Article Title: CD24-positive cells from normal adult mouse liver are hepatocyte progenitor cells.

doi: 10.1089/scd.2010.0352

Figure Lengend Snippet: FIG. 3. CD24 expression in normal, untreated, and DDC-treated liver. (A–F) Colocalization of CD24 and CK19 expression in wild-type untreated and DDC-treated livers. CD24 expression on bile duct epithelium was weak in untreated liver compared with red blood cells (A). Note that red blood cells in the vein (A) are strongly stained with CD24, but are CK19 negative in the merged view. To eliminate the high background signal from the red blood cells, the microscopic field was limited to the bile duct area outlined in yellow. The panel below each frame (A–C) shows staining of cells in ductular regions. CD24 (A, D, red) colocalized with CK19 (B, E, green) in the periportal zone in both untreated and DDC-treated liver (C, F). CD24 is induced in DDC-treated ductal cells (D), which correlated with the expansion of expression of CK19 (E). The magnification is 400·. (G–N) Immunohistochemical staining of CD24 and A6 antibodies on adjacent sections of normal wildtype and DDC-treated liver. In normal, untreated, wildtype liver, CD24 was expressed on red blood cells in the vein (labeled by lines) and ductal cells (G, H, green). In the same location of adjacent slides, A6 expression showed ductular staining similar to that of CD24 (I, J, green). In DDC-treated liver, the ductal expansion of CD24 expression (K, L, green) was similar to that of A6 (M, N, green). DNA is stained with DAPI blue. The magnification is 200·. FITC, fluorescein isothiocyanate. Color images available online at www.liebertonline.com/scd

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat IgG2a secondary antibody (Serotec; MCA278F, 1:100) was used to detect CK19 specifically.

Techniques: Expressing, Staining, Immunohistochemical staining, Labeling

Journal: Cell

Article Title: HIF Regulates Multiple Translated Endogenous Retroviruses: Implications for Cancer Immunotherapy

doi: 10.1016/j.cell.2025.01.046

Figure Lengend Snippet: Key resource table

Article Snippet: Mouse monoclonal IgG2 a AF647 , Santa Cruz Biotechnology , Cat#sc-24637, RRID: AB_737235.

Techniques: Virus, Recombinant, SYBR Green Assay, cDNA Synthesis, Purification, Sample Prep, Sequencing, Amplification, Software, Binding Assay, Transferring, Reverse Transcription, Low Protein Binding, Antibody Purification, Adjuvant, Enzyme-linked Immunospot

Journal: Radiation Oncology (London, England)

Article Title: Quantitative proteomic analysis reveals AK2 as potential biomarker for late normal tissue radiotoxicity

doi: 10.1186/s13014-019-1351-8

Figure Lengend Snippet: AK2 is differentially expressed between patients with and without grade ≥ 2 bf+. a Immunoblot analysis of AK2, ANX1, HSPA8 and IDH2 in control (0 Gy) and 8 Gy-irradiated T lymphocytes from the indicated patients. β-actin was used as loading control. b Quantification of the immunoblot presented in A using the ImageJ software. Data are the mean ± SEM; * p < 0.05 (2-tailed Mann-Whitney test). qRT-PCR analysis of AK2 and IDH2 mRNA expression in control (0 Gy) and 8 Gy-irradiated T lymphocytes from the same patients with and without grade ≥ 2 bf+. The graph shows the expression level in irradiated samples relative to non-irradiated samples

Article Snippet: Membranes were blocked with PBST (PBS plus 0.1% Tween-20) containing 5% non-fat milk at 25 °C for 60 min. Blots were then incubated (4 °C, overnight) with primary antibodies against adenylate kinase 2 (AK2) (1/100, sc-28,786; Santa Cruz Biotechnology), annexin A1 (ANXA1) (1/100, sc-11,387; Santa Cruz Biotechnology), galectin-1 (LSGAL1) (1/100, sc-19,277; Santa Cruz Biotechnology), heat shock cognate 71 kDa protein (HSPA8) (1/500, sc-7298; Santa Cruz Biotechnology) and isocitrate dehydrogenase 2 (IDH2) (1/200, sc-134,923; Santa Cruz Biotechnology).

Techniques: Western Blot, Control, Irradiation, Software, MANN-WHITNEY, Quantitative RT-PCR, Expressing

Journal: Human vaccines & immunotherapeutics

Article Title: The rLVS Δ capB / iglABC vaccine provides potent protection in Fischer rats against inhalational tularemia caused by various virulent Francisella tularensis strains.

doi: 10.1080/21645515.2023.2277083

Figure Lengend Snippet: Figure 5. Total serum IgG antibody in rats pre- and post-challenge (survivors) with Type A F. tularensis FRAN244/Schu S4 strain. Animals were immunized and challenged as described in the legend to Figure 2. Serum IgG antibody and subclasses specific to irradiated FRAN244 (Type A antigen) were assayed. a. Total serum antibody and subclasses in immunized rats prior to challenge with aerosolized F. tularensis Type A FRAN244/Schu S4 strain. For IgG, IgG2a, IgG2b, and IgG2c, all vaccinated groups had significantly greater titers than the PBS group (p<0.01-p<0.001). For IgG1, all vaccinated groups except LVS, rLVS 107 x1 rLVS 107 x2 groups had significantly greater titers than the PBS group (p <0.05 - p <0.001). Values represent median with interquartile range (Q1 and Q3 for the whiskers) of serum antibody for n = 7 or 8 per group. b. Total serum antibody and subclasses in immunized rats surviving challenge with aerosolized F. tularensis Type A strains. Values represent median with interquartile range (Q1 and Q3 for the whiskers) of serum antibody. Notes: in Fischer rats, IgG2b/2c indicates a Th1 type response and IgG1/2a indicates a Th2 type response. Values that are significantly different between two groups are marked with asterisk(s) over an open horizontal line crossing above the two groups. * p <0 .05; ** p < 0.01; and *** p < 0.001. Additional descriptive statistical values are included in Supplemental Tables S2–5.

Article Snippet: After the plates were washed as previously mentioned, diluted goat anti-rat-IgG, -IgG1, -IgG2a, -IgG2b, or -IgG2c horseradish peroxidase conjugate (1:5,000; Southern Biotechnology Associates, Inc.) was added to the wells and the plates were incubated for 30 min at 37°C.

Techniques: Irradiation

Journal: Human vaccines & immunotherapeutics

Article Title: The rLVS Δ capB / iglABC vaccine provides potent protection in Fischer rats against inhalational tularemia caused by various virulent Francisella tularensis strains.

doi: 10.1080/21645515.2023.2277083

Figure Lengend Snippet: Figure 6. Total serum IgG antibody in rats immunized with rLVS vaccines prior to and post challenge with Type A F. tularensis FRAN254 or Type B FRAN255 strain. Animals were immunized and challenged as described in the legend to Figure 3. Sera were collected in immunized rats prior to and after challenge with Type A or Type B strains and assayed for total IgG antibody specific to irradiated FRAN244 (Type A antigen) and FRAN255 (Type B antigen). (a). Prior to challenge; All vaccinated groups in Panel a had significantly greater titers than the PBS group (p <0.01-p <0.0001). (b). Post challenge (survivors only). Values represent median with interquartile range (Q1 and Q3 for the whiskers) of serum antibody titers for n = 6–8/group. * p < 0.05; ** p <0 .01; and *** p < 0.001. Additional descriptive statistical values are included in Supplemental Tables S6,7.

Article Snippet: After the plates were washed as previously mentioned, diluted goat anti-rat-IgG, -IgG1, -IgG2a, -IgG2b, or -IgG2c horseradish peroxidase conjugate (1:5,000; Southern Biotechnology Associates, Inc.) was added to the wells and the plates were incubated for 30 min at 37°C.

Techniques: Vaccines, Irradiation