Article Title: Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes
Figure Lengend Snippet: Characterization of SIDT2. (A) Lysosomes (Lys) were isolated from mouse brain homogenates (Hom), and analyzed by immunoblotting using polyclonal goat anti-SIDT2 antibody and antibodies against LAMP2 (lysosomal marker), RAB7A (late endosome and lysosome), RAB5A (early endosome), CANX (endoplasmic reticulum), COX4I1 (mitochondria), GOLGA1 (Golgi apparatus), GAPDH (cytosol), LMNA/lamin A (nuclei), and MAP1LC3A/B (autophagosome). (B) Neuro2a cells expressing GFP-tagged SIDT2 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified using ImageJ software (right panel, n = 3). ( C ) Neuro2a cells expressing GFP-tagged SIDT2 were fixed, and immunostained using anti-RAB7A, anti-EEA1 (early endosomal marker) or anti-MAP1LC3A/B antibodies. Fluorescent images were obtained using confocal microscopy. Scale bars: 5 μm. Colocalization rate was quantified (right panels, n = 3). (D) Lysosomes were isolated from HeLa cells expressing SIDT2-FLAG or CTSB-FLAG. Isolated lysosomes (4 μg protein) were incubated with the indicated concentrations of trypsin at 37°C for 5 min. Proteins in the samples were analyzed by immunoblotting using an anti-FLAG antibody. (E) LAMP2C and SIDT2 or SIDT2-FLAG were overexpressed in HeLa cells. Cell lysates were prepared and immunoprecipitated with an anti-FLAG antibody. Cell lysates and the resulting immunoprecipitant were analyzed by immunoblotting. (F) Lysates were prepared from HeLa cells overexpressing SIDT2 and LAMP2C or LAMP2C-FLAG and coimmunoprecipitation assays performed. (G) Endogenous interaction of SIDT2 with LAMP2C. Coimmunoprecipitation assays were performed using mouse brain lysates. (H) Neuro2a cells coexpressing FLAG-tagged LAMP2C and GFP-tagged SIDT2 were fixed, and immunostained using anti-FLAG antibody. Scale bars: 10 μm. Colocalization rate was quantified (right panel, n = 3).
Article Snippet: The primary antibodies used for immunoprecipitation or immunoblotting were: polyclonal goat anti-SIDT2 (N-20; Santa Cruz Biotechnology, sc-54151), polyclonal goat anti-SIDT1 (GeneTex, GTX88799), monoclonal rat anti-LAMP2 (M3/84; Santa Cruz Biotechnology, sc-19991), monoclonal mouse anti-DYKDDDDK (FLAG; Wako Pure Chemical Industries, 018-22783), monoclonal rabbit anti-SCARB2/LIMP2 (EPR12080; Abcam, ab176317), monoclonal mouse anti-RAB7A (Rab7-117; Abcam, ab50533), monoclonal mouse anti-RAB5A (RAB5A, member RAS oncogene family) (D-11; Santa Cruz Biotechnology, sc-46692), monoclonal mouse anti-MAP1LC3A/B (4E12; MBL, M152-3), polyclonal rabbit anti-LMNA (lamin A) (H-102; Santa Cruz Biotechnology, sc-20680), monoclonal mouse anti-COX4I1 (cytochrome c oxidase subunit IV isoform 1) (20E8C12; Life Technologies, A21348), monoclonal mouse anti-GOLGA1/Golgin-97 (CDF4; Life Technologies, A-21270), polyclonal rabbit anti-CANX (calnexin) (Abcam, ab22595), monoclonal mouse anti-GAPDH (6C5; Merck Millipore, CB1001), polyclonal rabbit anti-TUBB (tubulin, β)/β-tubulin (Cell Signaling Technology, 2146), and monoclonal mouse anti-ACTB/β-actin (AC-15; Sigma-Aldrich, A5441).
Techniques: Isolation, Marker, Expressing, Incubation, Fluorescence, Laser-Scanning Microscopy, Software, Confocal Microscopy, Immunoprecipitation