Mouse anti eea1

Protocols have been extensively described recently (Lucocq et al., 2001 (link), Karanasios and Ktistakis, 2015 (link)). In brief, cells on coverslips were fixed in 3.7% Formaldehyde, permeabilized in 0.1% NP40 and stained in a blocking solution containing fish gelation and 0.05% NP40. Antibodies used and their final dilution are as follows: rabbit anti-OPTN (Cayman Chemicals), 1:100; mouse anti-WIPI2 (Bio-Rad), 1:200; rabbit anti-phospho(S172)TBK1 (Cell Signalling), 1:50; mouse anti-Ubiquitin FK2 (Enzo), 1:100; rabbit anti-FIP200 (ProteinTech), 1:100; rabbit anti-ATG9 (Cell Signalling), 1:100; mouse anti-TOMM20 (Abcam), 1:100; rabbit anti-ATG13 (Sigma), 1:100; rabbit anti-LC3 (Sigma), 1:150; rabbit anti-NDP52 (GeneTex), 1:100; rabbit anti-Tax1BP1 (ProteinTech), 1:100; rabbit anti-VAPA (Sigma), 1:200; rabbit anti-VAPB (Sigma), 1:200; mouse anti-EEA1 (BD Biosciences), 1:70; mouse anti-LAMP2 (Developmental Studies Hybridoma Data Bank), 1:200; mouse anti-TUBULIN (Sigma), 1:300; mouse anti-cytochrome C (Abcam), 1:200; rabbit anti-phospho(S351)p62 (kind gift from Dr Masaaki Komatsu), 1:100; rabbit anti-TRAF2 (Abcam) 1:100.

HeLa cells were infected with either mCherry-expressing WT or Δstmp1 mutant bacteria in a 6-well plate for 2 h, washed extensively with PBS, and incubated in 10% RPMI. At 2 dpi, the infected cells were replated onto coverslips placed in a 24-well plate (5 × 10⁴ cells per well). The next day, cells were fixed in 2.5% PFA for 15 min and blocked/permeabilized for 20 min in 1% bovine serum albumin (BSA) and 0.1% saponin in PBS. Cells were then incubated with mouse anti-EEA1 (1:500; BD Biosciences) and rabbit anti-LAMP1 (1:1,000; Abcam) for 1 h followed by Alexa Fluor secondary antibodies (1:1,000) for 1 h. Following washing with PBS, coverslips were mounted using ProLong gold with DAPI and visualized on a Leica inverted DMI6000B microscope using a 63× oil immersion objective. Images were captured and processed identically, and the fluorescence intensity of EEA1 and LAMP1 was measured (ImageJ) and normalized to cell area, excluding the CCV from intensity measurements. At least 20 cells were measured per condition for each of three independent experiments.

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.

Mouse anti-FLAG (M2) and anti-α-tubulin monoclonal antibodies and rabbit anti-HA and anti-FLAG polyclonal antibodies were purchased from Sigma. Mouse anti-EEA1, anti-GM130, and anti-Rab11 monoclonal antibodies were purchased from BD Biosciences (Bedford, MA). Rabbit anti-TGN46 polyclonal antibody was purchased from Abcam (Cambridge, UK). Mouse anti-LAMP-1 monoclonal and Rabbit anti-Rab5 polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-HA (12CA5) and anti-FLNa monoclonal antibodies were purchased from Roche Applied Science (Indianapolis, IN) and Millipore (Billerica, MA), respectively. Secondary antibodies conjugated to Alexa Fluor 488 or 568, Alexa Fluor 568-phalloidin (Invitrogen), hoechst 33258 (Dojido laboratories, Kumamoto, Japan) were also purchased from commercial sources. Rabbit anti-FilGAP polyclonal antibody was prepared as described previously [9] (link). Rabbit anti-ARHGAP22 polyclonal antibody was directed against amino acid residues 469–485 (RGHRRASSGDRLKDSGS) of human ARHGAP22. The peptide was coupled through cysteine at the NH₂-terminal residue to keyhole limpet hemocyanin (KLH) and was used to raise the antiserum. The antiserum specific to ARHGAP22 was affinity-purified with the immobilized peptide.