Anti rfp

HeLa cells were lysed with lysis buffer (0.5% Triton X-100 in PBS) containing protease inhibitors (Roche) for 15 min on ice, after which the samples were centrifuged at 16,000 × g for 10 min at 4°C. The resulting supernatants were incubated for 16 hr while rotating at 4°C with 8 µg/ml anti-GFP antibody (Roche). Protein A/G PLUS-Agarose (Santa Cruz) was then added to the samples and incubated with rotation for 2 hr at 4°C. Immunoprecipitated fractions were washed two times with lysis buffer and two more times with PBS, and heated to 95°C for 5 min with ×1 Laemmli SDS sample buffer. The samples were subjected to SDS–PAGE and Western blotting with anti-GFP (Santa Cruz) and anti-RFP (Abcam) antibodies.

Western blotting analyses were carried out as previously reported.^{29 (link)} All antibodies in this study were used as follows: anti-β-tubulin (1:1000, Huaan, Hangzhou, China); anti-JWA (1:500, AbMax, Beijing, China); anti-GAPDH (anti-glyceraldehyde 3-phosphate dehydrogenase), anti-his, and anti-Flag (1:2000, Beyotime, Shanghai, China); anti-MARCH8 and anti-DR4 (1:1000, Proteintech, Chicago, IL, USA); anti-DR4 and anti-Ub (1:500, Santa Cruz, Dallas, TX, USA); anti-PARP-1 and anti-caspase-8, -9, -3 (1:1000, Cell Signaling Technology, Beverly, MA, USA); anti-RFP, anti-TNFR1, anti-Fas and anti-DR5 (1:2000, Abcam, Cambridge, UK). Cycloheximide (Sigma-Aldrich, St Louis, MO, USA) and Lseupeptin (Amresco, Solon, OH, USA) were used at the indicated concentrations.

Ovaries from recipient and control mice were fixed with the Tissue-Tek OCT Compound (Sakura Finetek Middle East, Dubai, United Arab Emirates) and sliced in 5 µm thick sections. Slides were washed twice with PBS and kept in blocking solution for 30 min at room temperature. Slides were then incubated with rabbit polyclonal anti-RFP (1∶200; Abcam) or anti-AMH (1∶200, AbD SeroTec) at 4°C overnight. Non-immune immunoglobulins of the same isotype as the primary antibody were used as negative controls. Cells or sections were washed three times with 1×PBS and probed with TRITC–labeled IgG (1∶200, Santa Cruz, CA, U.S.) or FITC-labeled IgG (1∶200, Santa Cruz, CA, U.S.). Fluorescence images were taken using a Leica DMI3000 microscope (Wetzlar, Germany).

The anesthetized mice were perfused with with 4% paraformaldehyde (PFA) dissolved in phosphate buffered saline (PBS), and isolated brains were post-fixed with 4% PFA at 4°C for over night. After washing with PBS, the brains were cryoprotected with 30% sucrose solution, and embedded in Tissue-Tek. The frozen brains were sectioned at a thickness of 35 μm using a Cryostat (CM1900 and CM1850, Leica), and incubated with primary antibodies, including anti-GFP (rabbit polyclonal, Life Technology or rat monoclonal, Nakarai Tesque), anti-RFP (rabbit polyclonal, Abcam), anti-GLAST (goat polyclonal, Frontier Institute), anti-GFAP (mouse monoclonal, SIGMA), anti-ß-gal (rabbit polyclonal, MP Biomedicals), anti-activated caspase3 (rabbit polyclonal, Cell signaling), and anti-S100 (rabbit polyclonal, DAKO). After washing, the sections were incubated with secondary antibodies, including Alexa-Fluor 488, 594 or 633-conjugated anti-rabbit, anti-mouse, anti-rat and anti-goat antibodies (Life technologies). Nuclear staining was performed with Hoechst 33258. Sections were analyzed with either a fluorescent microscope (BX51, OLYMPUS) equipped with a cooled CCD camera system (DP71, OLYMPUS) or a laser confocal microscope (FV1000D, OLYMPUS).

Stably transfected HK-2 cells expressing RFP-tagged cystinosin-LKG were grown in the 8 wells Chamber Slides (BD Falcon). Cells were fixed in 4% paraformaldehyde for 10 min at 4°C, permeabilized with 0.05% Triton X-100 for 5 min at 4°C, blocked with 1% FBS in PBS for 1 h at room temperature, and stained with primary antibody diluted 1:100 for 1 h at 4°C, followed by two 10 min washes with PBS and incubation with FITC-conjugated anti-mouse IgG antibody for 30 min at 4°C. Lysosomes were stained with anti-LAMP-2 or anti-LAMP-1 mAb (Santa Cruz Biotechnology), endoplasmic reticulum (ER) with anti-PDI mAb (Abcam), Golgi with anti-GM130 mAb (BD Biosciences).
HK-2 transfected with cystinosin-LKG and ΔSSLKG conjugated with RFP or V5His were fixed in 4% paraformaldehyde for 10 min at 4°C, permeabilized 30 min at room temperature with 0.05% saponin, 0.05% BSA, 50 mM NH₄Cl, blocked with 1% BSA in PBS for 1 h, and stained with anti-RFP (Abcam) or anti-His Tag, clone HIS.H8 (Millipore) diluted 1:200 for 1 h, incubated with FITC-conjugated anti-rabbit or anti-mouse IgG antibody respectively, in alternated washing steps with PBS.

Samples were suspended in the Laemmli buffer and resolved on SDS-PAGE using 4%–20% gradient gels (Bio-Rad, Hercules, CA, USA) at ∼5 mg/lane total protein. For immunoblot, proteins resolved on gels were transferred to nitrocellulose membranes (Bio-Rad) using Transblot (Bio-Rad). After incubating in blocking buffer (5% [w/v] nonfat dry milk, 1×Tris-buffered saline [TBS; 150 mM NaCl, 50 mM Tris–HCl, pH 7.5, 0.1% Tween-20]), membranes were first probed with primary antibodies diluted in 2.5% (w/v) nonfat dry milk, 1× TBS; anti-SYP132 (1:3,000, Agrisera), anti-AHA1 (1:3,000, Eurogentec), anti-RFP (1: 10,000; Abcam, Cambridge, UK), anti-GFP (1:10,000; Abcam), anti-H⁺-ATPase (1:10,000; Agrisera), anti-PR1 (1:10,000; Agrisera), anti-BIP (1:10,000; Agrisera), anti-HA (1:10,000; Roche, Basel, Switzerland), or anti-VP16 (1:10,000; Abcam). Subsequently, after washing with wash buffer (1× TBS), secondary antibody goat anti-rabbit-horseradish peroxidase conjugate (1:20,000; Abcam) was applied. Proteins were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and imaged by Fusion Chemiluminescence imager (Vilber, Marne-la-Vallée, France). Total proteins were visualized by staining the membrane with Coomassie or Ponceau. Band density was measured by densitometry using the ImageJ software.