Anti gfp antibody

Mouse liver tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in OCT, and cryosectioned at 4 μm thickness. Thereafter, sections were rinsed in PBS to remove the OCT, stained with DAPI, and sealed using mounting buffer (Sigma‐Aldrich). Cas9 and sgRNA expression was indicated by mCherry and GFP, respectively, under the microscope. For Western blot, liver tissue was homogenized in liquid nitrogen and the total protein content was released in lysis buffer (50 mM Tris‐base (pH 7.4), 150 mM NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% SDS, and Complete Mini Protease Inhibitor cocktail (Roche)). The expression of GFP and FIX protein was visualized by anti‐GFP antibody (Santa Cruz) and anti‐F9 polyclonal antibody (Protein tech). For qPCR, liver tissue was homogenized in liquid nitrogen and the total RNA was isolated with RNAiso Plus (TaKaRa). RT–PCR was performed with SYBR Green (TaKaRa). The primers for qPCR were listed in Appendix Table S6.

For pull-downs of overexpressed Arl3-Flag 2.5 × 10⁶ HEK293 cells were seeded in 15 cm² dishes 24 hr prior to transfection. Cells were transfected using Polyethylenimine (PEI) at a ratio 3:1 of PEI (µg) : total DNA (µg). Cells were induced to ciliate by withdrawing serum for 30 hr. ~2.5 × 10⁷ cells (1 × 15 cm² dish) were lysed in 1 ml lysis buffer for 30 min at 4°C. For pull-downs of endogenous Arl3 1 × 10⁸ cells (4 × 15 cm² dish) were used. Lysate was cleared by centrifugation and protein concentration normalized. Per sample 50 µg GST-PDE6δ was coupled to 50 µl glutathione agarose which was incubated with cleared lysates for 45 min at 4°C. Cleared lysate was removed and beads washed 2x with 500 µl buffer M. Samples were eluted with 1 × SDS-loading buffer. For the detection of affinity-precipitated endogenous Arl3 an anti-Arl3 antibody (Novus Biologicals) was used, and in case of Arl3-Flag an anti-Flag antibody (Thermo Scientific) was used. Expression of Arl13B-GFP was checked using an anti-GFP antibody (Santa Cruz Biotechnology) and antibody against S-peptide, which is located between Arl13B and GFP in pGLAP5. The level of Arl3·GTP was quantified using ImageJ. Experiments were repeated two or more times.

HIV-1 proviral constructs pNL4-3 [62] (link), and pYU2 [63] (link) along with zidovudine (AZT)were obtained from the NIH-ARRRP. Transfection of the dominant negative Rab11 mutant (DN Rab11, Addgene) was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's manual. Western blot analysis was carried out as previously described [64] (link). Colchicine, dynasore hydrate, DEAE-dextran hydrochloride and (E)-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL) were purchased from Sigma. Lysosomal inhibitors pepstatin A, leupeptin and E-64 were purchased from AG Scientific. anti-GFP antibody was purchased from Santa Cruz Biotechnology, Inc. The HIV-1 p24 monoclonal antibody [65] (link) was obtained from NIH-ARRRP. Transferrin was purchased from Invitrogen and staining was performed using the manufacturer's protocol.

Mouse renal epithelial Flp-In cells from the inner medullary collecting duct (IMCD3 Flp-In; kind gift from M.V. Nachury) were cultured at 37°C and 5% CO₂ in DMEM/F12, HEPES (Life Technologies) complemented with 10% fetal bovine serum (FBS), and 1% L-glutamine. Stable cell lines were generated as previously described (Sang et al., 2011 (link), Torres et al., 2009 (link)). In short, the parental IMCD3 Flp-In cell line contains a stably integrated FRT cassette and was co-transfected with pOG44 coding an FLP recombinase, and the appropriate construct cloned into pgLAP5 vector (Addgene), coding for a C-terminal S- and GFP-tag, using Lipofectamine 2000 (Life Technologies). Selection by supplementing the media with 200 μg/ml hygromycin (Merck) for successful stable genomic integration was carried out, and expression of the GFP fusion protein was checked by western blot using an anti-GFP antibody (1:500; Santa Cruz Biotechnology).