primary anti human rab7 monoclonal antibody d95f2  (Cell Signaling Technology Inc)

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Interaction and colocalization of the PEDV S protein with IFITM proteins. ( A ) Co-IP assays were performed to assess the interaction between the PEDV S1 protein and human or porcine IFITM proteins. HEK293T cells were cotransfected with plasmids encoding PEDV S1-Fc and various HA-tagged IFITM proteins. At 24 h post-transfection, the cells were harvested. Immunoprecipitation was conducted using an Fc tag antibody, and the presence of IFITM proteins was detected by Western blotting (IB: Anti-HA). Whole-cell lysates (WCLs) were analyzed to confirm expression levels (IB: Anti-Fc, Anti-HA, Anti-β-Tubulin). The Co-IP was repeated three times, yielding similar results. ( B ) Confocal microscopy images showing the intracellular localization of porcine IFITM1 in LLC-PK1 cells are presented. IFITM1 (red) colocalizes with clathrin (green), EEA1 (green), Rab7 (green), and LAMP7 (green), as indicated in the merged images. DAPI (blue) was used to stain the nuclei. ( C ) Confocal microscopy images demonstrating the colocalization of the PEDV S protein (red) with HA-IFITM1 (green) in LLC-PK1 cells are shown. The merged image shows the nuclei stained with DAPI (blue).

Article Snippet: Additional antibodies included EEA1 mouse monoclonal antibody (cat#: 68065-1-Ig), HA tag mouse monoclonal antibody (cat#: 66006-2-Ig), Rab7 (D95F2) XP rabbit monoclonal antibody (cat#: 9367s), and CLTC monoclonal antibody (cat#: 66487-1-Ig) from Proteintech.

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Expressing, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: A Novel TRPC6 Mutation Causes Autosomal Dominant FSGS

doi: 10.1101/2025.02.11.637765

Figure Lengend Snippet: iPS-cell derived podocytes have reduced expression of slit diaphragm proteins, ER congestion, and reduced lysosomes. Panel A shows representative immunoblots of podocyte markers including Nephrin, Synaptopodin, TRPC6, Podocin, CD2AP, GLEPP1, and WT1, the housekeeping protein GAPDH. Panel B shows the quantification of immunoblots for the podocyte markers. Data represents three biologically independent experiments and is defined as ± standard error of the mean. We performed one-way ANOVA with Dunnett’s multiple comparison post hoc analyses with a family-wise alpha threshold of 0.05. Panel C shows electron micrograph of differentiated podocytes with protein aggresomes in the ER of Duke 5 and Duke 7 podocytes. Panel D shows LAMP1 vesicles, Podocin, and TRPC6 expression in podocytes. The LAMP1 vesicle number remains unchanged in the podocytes. Panel E shows lysosomal biogenesis-associated RAB7 expression in the podocytes. RAB7 expression was reduced in the mutants. Data is representative of three biologically independent experiments and the data is defined as ± standard error of the mean. We performed one-way ANOVA with Sidak’s multiple comparison post hoc analyses with a family-wise alpha threshold of 0.05. Scale bar: Panel C, top row 1000 µm, bottom row 500 nm; Panel D, top row 20 µm, bottom row 5 µm; Panel E, 100 µm.

Article Snippet: The primary antibodies used included guinea pig Anti-Nephrin (ARP; #GPN02, 1:500), rabbit Anti-Podocin (Abcam, mAb #ab50339, 1:1000), rabbit Anti-TRPC6 (Abcam, pAb #ab228771) and mouse Anti-CD2AP (B-4) (Santa Cruz, #sc-25272), Calnexin (C5C9) Anti-Rabbit (Cell Signaling, mAb #2679), LAMP1 (D2D11) XP Anti-Rabbit (Cell Signaling, mAb #9091), RAB7 (D95F2) XP and Anti-Rabbit mAb (Cell Signaling, mAb #9367).

Techniques: Derivative Assay, Expressing, Western Blot, Comparison

Journal: Cell reports

Article Title: Molecular determinants of the crosstalk between endosomal microautophagy and chaperone-mediated autophagy

doi: 10.1016/j.celrep.2023.113529

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The dilutions and sources of antibodies used for immunoblot (IB), immunoprecipitation (IP), cell immunofluorescence (cell-IF) and isolated LE/MVB immunofluorescence (LE-IF) in this study were as follows: (dilutions, commercial source, and catalog number indicated in parentheses): rabbit anti-aldolase (1:1000 (IB), Cell Signaling, 3188S), rabbit anti-Alix (1:500 (IB), Novus, NBP1–90201), mouse anti-Alix (3A9) (1:100 (LE-IF), Cell Signaling, 2171S), mouse anti-α synuclein (211) (1:2000 (IB), Santa Cruz, sc-12767), rabbit anti-ATP6V1B (1:1000 (IB), Abcam, ab200839), rabbit anti-Bag1 (1:1000 (IB), Abcam, ab32109), rabbit anti-Bag3 (1:1000 (IB), Novus, NBP2–27398SS), sheep anti-Bag6 (1:200 (IB) and 1:100 (LE-IF), R&D Systems, AF6438), rabbit anti-Bag6 (1:1000 (IB), 1:100 (LE-IF), 1μg (IP), Abcam, ab137076). rabbit anti-Cathepsin B (1:1000 (IB), Abcam, ab214428), mouse anti-cathepsin D (D-7) (1:500 (IB), Santa Cruz, sc-377299), rabbit anti-cathepsin L (H-80) (1:1000 (IB), Santa Cruz, sc-10778), rabbit anti-CHIP (C3B6) (1:1000 (IB), Cell Signaling, 2080), rabbit anti-Cyclophilin A (1:1000 (IB), Cell Signaling, 2175), mouse anti-EEA1 (1:1000 (IB), BD Biosciences, 610456, RRID AB_397830), rabbit anti-GAPDH (1:2000 (IB), Cell Signaling, 2118S), rabbit anti-GBA (1:1000 (IB), Sigma, G4171), rabbit anti-hexokinase (1:1000 (IB), Cell Signaling, 2024S), rabbit anti-HIP (1:1000 (IB), Stressgen, adi-spa-766), rabbit anti-HOP (1:1000 (IB), Enzo, adi-sra-1500), mouse IgM anti-Hsc70 (13D3) (1:3000 (IB) and 1:200 (LE-IF), Novus, nb120–2788), rabbit anti-hsp40 (1:1000 (IB), Assay Designs, adi-spa-410), rabbit anti-hsp90 (1:1000 (IB), Stressgen, ADI-SPA-835-F), rat anti-LAMP-1 (1D4B) (1:3000 (IB) 1:200 (LE-IF) 1:2000 (Cell-IF), Hybridoma Bank, 1D4B, RRID AB_2134500), mouse-anti LAMP-1 (LyC16) (1:1000 (IB) and 1:100 (LE-IF), Enzo, adi-vam-en001), rabbit anti-LAMP-2A (1:5000 (IB), ThermoFisher Scientific, 512200, RRID AB_2533900) rabbit anti-LC3 (1:1000 (IB), Cell Signaling, 2775, RRID AB_915950), rabbit anti-LRRK2 (MJFF3) (1:1000 (IB), Abcam, ab133475), rabbit anti-mTOR (1:2000 (IB), Cell Signaling, 2972), mouse anti-p62 (1:1000 (IB), Abcam, ab56416), rabbit anti-Rab7 (D95F2) (1:500 (IB), Cell Signaling, 9367), mouse anti-total Tau (DA9) (1:3000 (IB) and 1:200 (LE-IF), gift from Dr. Peter Davies), rabbit anti-Tsg101 (1:1000 (IB) and 1:100 (LE-IF), Abcam, ab30871), mouse anti-tubulin (1:1000 (IB), Sigma, T7451), rabbit anti-ubiquitin (1:1000 for IB and for Cell-IF, Dako, Z0458, RRID AB_2315524), mouse anti-Vps4 (E-8) (1:500 (IB) and 1:100 (LE-IF), Santa Cruz, sc-133122), rabbit anti-Hsc70 (1μg (IP), Enzo, ADI-SPA-757), rabbit normal IgG (1μg (IP), Cell Signaling, 2729S).

Techniques: Recombinant, Injection, Western Blot, Cell Culture, Stripping Membranes, Quantitative Proteomics, Multiplex sample analysis, Plasmid Preparation, shRNA, Software, Fluorescence, Microscopy