Rabbit anti calnexin

The following primary antibodies and dilutions were used for immunofluorescence
studies: mouse anti-Flag (Sigma-Aldrich, F3165) (1:1000), rabbit anti-TOM20 (Santa-Cruz,
sc11415) (1:1000), rabbit anti-PMP70 (Sigma-Aldrich, SAB4200181) (1:1000), mouse
anti-p230 (BD Biosciences, 611281) (1:1000), rabbit anti-Rab7 (Abcam, ab137029)
(1:1000), rat anti-Calnexin (Biolegend, 699401) (1:1000) and mouse anti-Neurofilament H
(Biolegend, 835801). Donkey anti-mouse, Goat anti-mouse IgG1, Goat anti-mouse IgG2a,
Goat anti-rabbit and Goat anti-rat, Alexa Fluor 488, 568 or 647 were used as secondary
antibodies (1:1000) (Invitrogen).
The following primary antibodies and dilutions were used for immunoblot analysis: mouse
anti-Flag (Sigma-Aldrich, F3165) (1:1000), mouse anti-VDAC1 (Abcam, ab14734) (1:1000),
rabbit anti-TMEM63C (Abcam, ab203486) (1:500), rabbit anti-Pex14 (Proteintech,
10594-1-AP) (1:1000), rabbit anti-Calnexin (Proteintech, 10427-1-AP) (1:1000), mouse
anti-Tubulin (Santa-Cruz, sc23948) (1:1000), rabbit anti-VAPB (Atlas, HPA013144) (1:500)
and mouse anti-ACSL4 (Santa-Cruz, sc-365230) (1:1000). Horseradish peroxidase-conjugated
anti-rabbit and anti-mouse IgG (GE Healthcare) were used as secondary antibodies
(1:3000).

NIH3T3 or MEFs were seeded on glasses and cultured for 24 hours prior to transfection with Fugene HD (Promega). The cells were allowed to recover for 24 to 36 hours. To visualize ciliary proteins, transfected cells were serum-starved in DMEM containing 0.5% FBS for 24 hours before other treatments. Fixation was done in 4% paraformaldehyde for 10 minutes at room temperature, permeablized with 0.3% Triton X-100 in PBS for another 10 minutes, followed by standard procedures for immunostaining. Primary antibodies used were rabbit anti-IFT88 (1:100; Proteintech), goat anti-SEC13 (1:50; Santa Cruz Biotechnology, Inc.), rabbit anti-Calnexin (1:100, Proteintech), rabbit anti-Clathrin heavy chain (1:200; Cell Signaling Technology (Danvers, MA)), rat anti-HA (1:300; Roche), mouse anti-acetylated Tubulin (1:1000; Sigma), rabbit anti-Gli3 (1:500; R&D (Minneapolis, MN)). Alexa-coupled secondary antibodies were purchased from Life Technologies Corp. Fluorescence images were captured using a laser scanning confocal microscope (LSM 710; Carl Zeiss) with a 63 × 1.4 numerical aperture (NA) oil objective and images were analyzed using software Zeiss ZEN 2011. Colocalization was quantified by software Image-pro as described^{52 (link)}.

The exosomal and cellular proteins were extracted by DNA/RNA/protein Isolation Kit (catalog number: R6734-02, OMEGA). The sample was denatured by heating, separated by SDS-PAGE and transferred to a PVDF (polyvinylidene fluoride) membrane. Next, the membranes were blocked with 5% skimmed milk powder and separately probed with rabbit anti TSG101 (catalog number: GB11618, servicebio), rabbit anti HSP70 (catalog number: GB11241, servicebio), rabbit anti TSC1 (catalog number: GB11882, servicebio), rabbit anti calnexin (catalog number: 10427–2-AP, Proteintech), rabbit anti GAPDH (catalog number: GB11002, servicebio), rabbit anti β-actin (catalog number: GB11001, servicebio) and mouse anti DKK3(catalog number: 66758–1-Ig, Proteintech) overnight at 4 °C with a final dilution 1:1000 (v/v) in 5% milk. After three times of washing, the membranes were incubated with goat anti-rabbit secondary antibodies (GB23303, Sevicebio, Wuhan, China) or goat anti-mouse secondary antibodies (GB23301, Sevicebio, Wuhan, China) with 1:2000 dilution (v/v) at 37 °C for 1.5 h. The images of membranes treated with ECL (enhance chemiluminescence) were captured by Western Blotting Detection System (Tiangen, Beijing, China).