Mouse anti lamp1

Slides containing 5 μm tissue sections were deparaffinized followed by antigen unmasking in sodium citrate buffer. Then, the tissue sections were incubated with 1% bovine serum albumin (room temperature for 30 min) to block non-specific antibody binding. Following this, the samples were incubated at 4°C overnight with the following primary antibodies: rabbit anti-LC3 (1:100 dilution) (12741-Cell Signaling Technology, Danvers, Massachusetts, USA) and mouse anti-LAMP1 (1:100 dilution) (15665- Cell Signaling Technology, Danvers, Massachusetts, USA). Then, after washing with 0.1M PBS (pH 7.4), samples were incubated with Alexa Fluor-488-conjugated goat anti-rabbit IgG (1:100 dilution; catalog no. RBaf48801; Fcmacs, Nanjing, China) and Tritc-conjugated goat anti-mouse IgG (1:100 dilution; catalog no. BA1089; Boster, Wuhan, China), secondary antibodies for two hours at 37°C. Nuclei were counterstained with 4′,6-diamidino-2- phenylindole (DAPI) (catalog no. 13G04A76; Boster, Wuhan, China). Visualization of fluorescence on sections were observed under an Olympus BX53 microscope and fluorescent images were captured with an Olympus DP73 digital color camera

The following antibodies were used: mouse anti-ubiquitin (Santa Cruz(P4D1), sc-8017, 1:1000); rabbit anti-LC3 (Cell Signaling Technology (D11), #3868, 1:1000); mouse anti-LAMP1 (Cell Signaling Technology (D4O1S), #15665, 1:200); mouse anti–USP8 (Sigma-Aldrich (US872), SAB4200527, 1:1000); rabbit anti-EPG5 (Beiijng TDY Biotech CO., Ltd, TDY349F, 1:1000); mouse anti-actin (Sigma-Aldrich (AC-15), A5441, 1:5000); rabbit anti-HA tag (Abcam, ab9110, 1:1000); mouse anti-FLAG tag (Abmart, M20008, 1:1000); rabbit anti-Myc tag (Abmart, M20002, 1:1000); Alexa Fluor^® 488 donkey anti-rabbit IgG (H + L) (Invitrogen Thermo Fisher Scientific, A21206, 1:500); Alexa Fluor^® 555 donkey anti-mouse IgG (H + L) (Invitrogen Thermo Fisher Scientific, A21203,1: 200); Alexa Fluor^® 647 donkey anti-mouse IgG (H + L) (Invitrogen Thermo Fisher Scientific, A21235, 1:500). Chloroquine, Hoechst 33342, Baf-A1, and MG132 were obtained from Sigma-Aldrich.

For immunofluorescence (IF) of HEPG2 cells, 100,000 cells were grown on coverslips in 12-well plates and incubated with RSPO2-flag conditioned media for 1.5 h. For H1581 cells, 150,000 cells were grown on coverslips in 12-well plates and transfected with 50 nM siRNA for 48 h. HEPG2 and H1581 cells were fixed in 4% PFA for 15 min, blocked and permeabilized with 5% BSA in PBSTB for 1 h and treated with 1:250 diluted rabbit anti-FGFR4 (Cell Signaling Technology 8562 S), mouse anti-EEA1 (BD 610457), mouse anti-clathrin (BD 610499), rabbit anti-FGFR1 (Cell Signaling Technology 9740 S), mouse anti-Flag (Sigma F3156) and mouse anti-Lamp1 (Cell Signaling Technology 15665 S) overnight at 4 °C. 1:500 diluted donkey anti-mouse Alexa Fluor 647 (Invitrogen A31571), donkey anti-rat Alexa Fluor 488 (Invitrogen A21208), donkey anti-rabbit Alexa Fluor 546 (Invitrogen A10036) and goat anti-mouse Alexa Fluor 488 (Invitrogen A11029) and Hoechst dye (1:500) were applied for 2 h at room temperature. Quantification of IF was performed using FIJI (Image J) v1.51k software. Tyramide Signal Amplification to detect RSPO2-HRP was executed as previously described^{32 (link)}. Representative images were obtained using LSM 700 (Zeiss) and processed with Zeiss ZEN 2012 (black edition) version 2.5.