Journal: The Journal of Cell Biology

Article Title: Endoplasmic reticulum patterns insect cuticle nanostructure

doi: 10.1083/jcb.202503127

Figure Lengend Snippet: The role of Ref(2)P in Gox localization and ubiquitination. (A) Ref(2)P is specifically enriched in the shaft region of olf hair cells, which is labeled by HA–Gox. Maxillary palp 42 h APF. (B) Subcortical localization of Ref(2) in control and gox mutant olf hair cells, and their quantification. Line intensity profiles were obtained from 5–7 individual hairs per genotype using the PlotProfile function in Fiji and averaged after normalization with custom Python scripts. Gray line: Ref(2)P distribution in control; magenta line: Ref(2)P in gox KO. Error shading represents mean ± SD. (C) Subcortical Gox localization was lost in Ref(2)P RNAi. Right graph: quantification. Right panel: quantification of fluorescence intensity profiles. Gray line: HA–Gox in control; magenta line: HA–Gox in Ref(2)P knockdown cells. No formal statistical test was applied because the plots represent averaged fluorescence intensity distributions (D) Interaction of Gox2 with Ref(2)P and ubiquitination in S2 cells. Gox–Flag and Ref(2)P-Myc were co-expressed and immunoprecipitated with the tag antibodies. Immunoprecipitate of Ref(2)P containing a low molecular weight form of Gox (*, lane 4). The increased amount of high molecular weight forms of Gox was induced by Ref(2)P (compare lane 7 and 8). Note that cross-reactivity of HRP-conjugated antibodies to the IgG (marked with **) used for immunoprecipitation. (E) Properties of Gox8KR. Gox8KR (*) and Ref(2)P (**) were co-immunoprecipitated with each other (lane 8 and 12). *** indicates the band of Ref(2). Although Ref(2) caused a slight increase in Gox amount and molecular mass (lane 16), no specific increase in ubiquitination level was observed (lane 20). (F) WT gox and gox8KR were expressed by neur-Gal4. (G) Line intensity profiles showing the distribution of Flag (pink) and ER marker tdTomato–Sec61b (gray) in olf hair cells expressing UAS–Gox–Flag (top) or UAS–Gox8KR–Flag (bottom). Profiles were obtained and averaged as in B and C. No formal statistical test was applied; error shading represents mean ± SD. (H) AlphaFold 2 model of Gox/Osi23 (pLFFT score 68.12), and the location of the two mapped ubiquitinated lysine (K101 and K131) identified by the LC-MS/MS. Alpha helices are labeled (H1–H6). Molecular weight markers (kDa) are indicated for all blots, and uncropped images are provided in SourceData FS4.pdf.

Article Snippet: The FlexAble CoraLite Plus 488 Antibody Labeling Kit for Mouse IgG1 was used to label the mouse anti-Cnx antibody for double labeling with two mouse antibodies.

Techniques: Ubiquitin Proteomics, Labeling, Control, Mutagenesis, Fluorescence, Knockdown, Immunoprecipitation, Molecular Weight, High Molecular Weight, Marker, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: Scientific reports

Article Title: Graft cell expansion from hiPSC-RPE strip after transplantation in primate eyes with or without RPE damage.

doi: 10.1038/s41598-024-60895-w

Figure Lengend Snippet: Figure 2. Temporal changes in the RPE layer following micropulse laser treatment. (A) Retinal photographs of the eyes 2 and 5 days after the application of the subthreshold micropulse laser. (B) Autofluorescence images 2 and 5 days after the subthreshold microsecond pulse laser treatment. (C) Optical coherence tomography (OCT) images of the coagulated site 2 and 5 days after the microsecond pulse laser treatment. The yellow line indicates the locus of laser treatment. (D) Hematoxylin and eosin staining of the retinal pigment epithelium (RPE) damaged site immediately after and 2 and 5 days after the microsecond pulse laser treatment. (E) Immunostaining of RPE65 and rhodopsin at intact and RPE-damaged areas immediately after and 2 days after the microsecond pulse laser treatment. Yellow line indicates the locus of laser treatment. ONL outer nuclear layer. Scale bars: (D) 100 μm, (E) 500 μm.

Article Snippet: For the co-immunostaining using two different mouse antibodies for Ret P1 and RPE65, the FlexAble CoraLite®488 antibody labeling kit for mouse IgG1 (Proteintech KFA021) was used according to the manufacturer’s instructions.

Techniques: Tomography, Staining, Immunostaining

Journal: Scientific reports

Article Title: Graft cell expansion from hiPSC-RPE strip after transplantation in primate eyes with or without RPE damage.

doi: 10.1038/s41598-024-60895-w

Figure Lengend Snippet: Figure 4. Histology of hiPSC-RPE strip transplant at an RPE-damaged site (M4). (A) Low magnified hematoxylin and eosin (HE) image and the immunostaining image of the neighboring section with anti-human MERTK (hMERTK) and − RPE65 antibody in the hiPSC-RPE strip transplanted area over RPE damage. Yellow line indicates MERTK positive grafted area. (B) Immunostaining image of hMERTK and rhodopsin. Yellow line indicates hMERTK positive grafted area. (C) Engrafted RPE cells are positive for STEM121 and hMERTK on the apical side (arrows) while host RPE cells at the border show only autofluorescence but not STEM121 or hMERTK positivity (C’). (D) hMERTK on the apical side of transplanted RPE cells (arrows) face and interact with rhodopsin-positive outer segments of host photoreceptors. (E) Immunohistochemistry of type IV collagen in intact and RPE-damaged sites at 2 days after laser treatment. Expression of type IV collagen is disrupted after microsecond pulse laser treatment. (F) Expression analysis of type IV collagen in the intact, laser-damaged, and transplant sites. Expression of type IV collagen is identified at the basement membrane of transplanted RPE. (G) Expression analysis of type IV collagen at the basement membrane of hMERTK-positive RPE cells. Magnified view of yellow boxed part on the right. ONL outer nuclear layer, INL inner nuclear layer. Scale bars: (A) 200 μm, (B) 100 μm, (C,D) 10 μm, (E) 50 μm, (F) 100 μm, (G) 50 μm.

Article Snippet: For the co-immunostaining using two different mouse antibodies for Ret P1 and RPE65, the FlexAble CoraLite®488 antibody labeling kit for mouse IgG1 (Proteintech KFA021) was used according to the manufacturer’s instructions.

Techniques: Stripping Membranes, Immunostaining, Immunohistochemistry, Expressing, Membrane

Journal: The Journal of biological chemistry

Article Title: Human V-ATPase a-subunit isoforms bind specifically to distinct phosphoinositide phospholipids.

doi: 10.1016/j.jbc.2023.105473

Figure Lengend Snippet: Figure 9. Localization of expressed a2NT-GFP in mouse 4T1 breast cancer cells. A, micrographs of 4T1 cell expressing WT a2NT-GFP probed with anti- GFP antibody (green) and anti-TGN46 antibody (red) in indirect immunofluorescence microscopy. B, micrograph of 4T1 cell expressing the a2NT-GFP containing the K221A mutation, probed as described in A. Both A and B are single images from near the center of a Z-stack, selected because as having a readily visible nucleus.

Article Snippet: The anti-TGN46 primary antibody (Proteintech 66477) was labeled with FlexAble CoraLite Plus 647 for mouse IgG1 and used at a 1:500 dilution.

Techniques: Expressing, Microscopy, Mutagenesis