rabbit polyclonal anti flotilin  (Novus Biologicals)

Journal: bioRxiv

Article Title: The Rubicon-WIPI axis regulates exosome biogenesis during aging

doi: 10.1101/2024.05.08.593233

Figure Lengend Snippet: (a) Immunoblotting of CD63 and Flotilin-1 (Flot-1) in the exosome fractions isolated from the culture medium of human mesenchymal stem cells (hMSCs) with knockdown of the indicated genes. The loading amount for each sample was set as follows: half of the exosome sample collected from 1 ml of culture medium. (b) Quantification of the exosomal CD63 levels in the hMSCs with knockdown of the genes indicated in (a). Bars represent means ± SEM. n = 4 independent experiments. *p < 0.05 (each vs control) by one-way ANOVA followed by Dunnett’s test. (c) Immunoblotting of the indicated proteins in the exosome fractions and the WCLs obtained from control and Rubicon -knockout (KO) mouse embryo fibroblasts (MEFs). The loading amount for each sample was set as follows: half of the exosome sample collected from 1 ml of culture medium, and 6 µg for the WCL. (d) Quantification of the exosomal levels of CD63, ALIX, and Flot-1 in (c). Bars represent means ± SEM. n = 3 independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001 by a two-tailed Student’s t-test. (e) Immunoblotting of CD63 and ALIX in the exosome fractions isolated from the cultured medium of control and Rubicon-overexpressing (OE) MEFs. The loading amount for each sample was set as follows: half of the exosome sample collected from 1 ml of culture medium. (f) Quantification of the exosomal CD63 and ALIX levels in (e). Bars represent means ± SEM. n = 4 independent experiments. *p < 0.05 by a two-tailed Student’s t-test. (g) Nanoparticle tracking analysis (NTA) of the extracellular vesicles (EVs) purified from the cultured medium of control or Rubicon -KO MEFs using a phosphatidylserine affinity magnetic resin (PS-affinity kit). (h) Quantification of the total particle concentration of the EVs in (g). Bars represent means. n = 6 independent experiments. ****p < 0.0001 by a two-tailed Student’s t-test. (i) Immunofluorescent images of CD63 (magenta) and DAPI (blue) in MEFs stably expressing GFP-Rubicon treated with 0.5 μM Apilimod for 1 hour. Scale bars, 50 μm.

Article Snippet: The following antibodies were used for western blotting at the indicated dilutions: anti-CD63 (MBL, D263-3, 1:5000; Abcam, ab134045, 1:2000), anti–Flotilin-1 (BD Biosciences, 610821, 1:10000), anti-ALIX (BioLegend, 634502, 1:1000), anti-Calnexin (Sigma-Aldrich, HPA009433, 1:1000), anti-Rubicon (Cell Signaling Technology, 8465, 1:2000), anti–α-Tubulin (PM054, MBL, 1:20000), anti-WIPI2 (Sigma-Aldrich, SAB4200400, 1:2000), anti-HRS (GeneTex, GTX101718, 1:1000), anti-VPS4 (Sigma-Aldrich, SAB4200025, 1:1000), anti-GFP (Cell Signaling Technology, 2555, 1:1000), anti-p21 (Abcam, ab109199, 1:1000), anti-γH2AX (Abcam, ab2893, 1:1000), anti-p16 INK4a (IBL, 11104, l g/ml), anti-LC3 (MBL, PM036, 1:2000), anti-Atg12 (Cell Signaling Technology, 2011S, 1:1000), anti-FIP200 (Proteintech, 17250-1-AP, 1:1000), anti-Atg2A (MBL, PD041, 1:1000), anti-Atg2B (Sigma-Aldrich, HPA019665, 1:1000), anti-Atg14 (Cell Signaling Technology, 96752S, 1:1000), anti–Beclin-1 (Cell Signaling Technology, 3738S, 1:1000), anti-UVRAG (MBL, M160-3, 1:2000), anti-ATG16L1 (Cell Signaling Technology, 8089, 1:1000), anti-PIK3C3 (Cell Signaling Technology, 3811, 1:1000), anti-WIPI1 (Sigma-Aldrich, W4769, 1:1000), anti-WIPI3 (Thermo Fisher Scientific, PA5-50864, 1:1000), anti-WIPI4 (Proteintech, 19194-1-AP, 1:1000), anti–β-Actin (MBL, M177-3.

Techniques: Western Blot, Isolation, Knock-Out, Two Tailed Test, Cell Culture, Purification, Concentration Assay, Stable Transfection, Expressing