Anti rfp

Samples were anesthetized (primary polyps) and fixed in 4% PFA in PBT for 6 h, washed in PBT and blocked in blocking buffer (80% PBT, 20% sheep serum, 1% bovine serum albuminum) for 2 h at room temperature. They were then incubated in primary antibody (rat, anti-RFP; Chromotek) 1:400 in blocking buffer overnight at 4°C, subsequently washed in PBT and incubated in secondary antibody (goat, anti-rat, DyLight 549; Jackson) 1:500 in blocking buffer containing phalloidin (3 μl/100 μl) for 3 h at room temperature. After an additional washing step, samples were mounted on glass slides in Vectashield.

Proteins from the sample lysate were fractionated by SDS–PAGE. The separated proteins were transferred from the gel to an Immobilon-PSQ polyvinylidene difluoride membrane (pretreated with methanol for 15 s; Millipore) using a transfer buffer (20 mM Tris, 150 mM glycine). The membrane was then blocked using PBS(pH 7.4) containing 3% non-fat dry milk for 30 min at room temperature with 50 r.p.m. shaking, followed by one wash with PBST (PBS with 0.1% Tween 20). Anti-Flag (1:2,000; #F3165; Sigma-Aldrich) and anti-BiP (1:1,000; #sc-33757; Santa Cruz Biotechnology), anti-GFP (1:2,000; #M20004; Abmart), anti-RFP (1:1,000; #5f8; Chromotek), anti-HA (1:2,000; #M20003; Abmart), anti-myc (1:2,000; #M20002; Abmart) and anti-actin (1:2,000; #M20009; Abmart) antibodies were added to PBSTM (PBS with 0.1% Tween 20 and 3% non-fat dry milk) and incubated at room temperature for 90 min, followed by three washes (5 min each) with PBST. The membrane was then incubated with a goat anti-mouse IRDye 800CW antibody (Odyssey, no. 926-32210; Li-Cor) at a ratio of 1:10,000 in PBSTM at room temperature for 30 min with 50 r.p.m. shaking. The membrane was washed three times (5 min each) with PBST, once for 5 min with PBS, and then visualized by excitation at 780 and 800 nm. Full-size images are presented in Supplementary Fig. 14.

Cells were seeded on gelatin-coated coverslips in 24-well plates and were transfected the next day. Cells were fixed 24 h later in fresh 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with PBS/BSA/Triton/Azide buffer (PBTA) [3% (wt/vol) BSA, 0.25% Triton, 0.01% NaN₃] for 1 h, and incubated overnight with primary antibodies (in dilution 1:500) in PBTA at 4 °C. The next day, the coverslips were washed in PBS and incubated with secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen A11001) or/and Alexa Fluor 594 (Invitrogen A11058) (all in dilution 1:1000), washed with PBS [in case of samples containing FLAG-DVL3 WT and derived variants stained with DAPI (1:5000)], and all coverslips were mounted on microscopic slides. Cells were then visualized on an Olympus IX51 fluorescent microscope using ×40 air or ×100 oil objectives and/or an Olympus Fluoview 500 confocal laser scanning microscope IX71 using ×100  oil objective. One hundred positive cells per experiment (n = 3) were analyzed and scored according to their phenotype into two/three categories (punctae/even); or non-recruited/partially/fully recruited for FZD6-dependent recruitment of DVL3. The antibodies used were as follows: anti-FLAG M2 (Sigma-Aldrich, #F1804), anti-DVL3 (Santa Cruz, #sc-8027), anti-CK1ɛ (Santa Cruz, #sc-6471), anti-RFP (Chromotek, #5h9) and anti-GFP (Fitzgerald, #20R-GR-011).

1 day-old adult animals were dissected for germline and freeze cracked on poly-lysine coated microscope slides. Dissected germlines were fixed in −20 °C methanol for 20 min. Fixed samples were washed with PBS-T (PBS supplemented with 1% tween-20) prior to primary antibody addition. Primary antibodies were incubated with the samples at 4 °C for overnight. Secondary antibodies were incubated at 37 °C for 1 h in the dark. Antibodies used: anti-PRG-1 (Custom, 1:1000); anti-mouse GFP (Thermofisher, A-11120; 1:400); OIC1D4 for PGL-1 staining (Developmental Studies Hybridoma Bank; 1:50); anti-RFP (Chromotek, clone 5F8). All fluorescence secondary antibodies were from Invitrogen and used at 1:500. Dissected and stained germlines were mounted with Vectorshield antifading agent supplemented with DAPI.

SDS-PAGE was performed with either 8% or 12.5% acrylamide gels. For the subsequent Western blotting, 0.2 μm nitrocellulose membranes (Advantec, Toyo Roshi Kaisha Ltd.) were used. The membranes were blocked in PBS containing 5% fat-free milk powder, 0.1% Tween-20 and 5 mM NaN₃ for at least 30 min. Incubation with the primary antibodies, all diluted in PBS containing 5% BSA and 0.1% Tween-20, was performed overnight at 4 °C. Primary antibodies used for this study were: anti-HA (Roche, 1:3000, 11867423001), anti-RGS-His (Qiagen, 1:2000, 34610), anti-Myc (ChromoTek, 1:1000, AB_2631398), anti-Vinculin (Sigma, 1:2000, V9264), anti-α-tubulin (Merck, 1:1000, TAT-1 00020911), anti-GFP (ChromoTek, 1:1000, 3H9), anti-RFP (ChromoTek, 1:1000, 6G6). The secondary antibodies were: HRP-antimouse IgG (Dako, 1:5000, P0260), HRP-antirat IgG (Thermo Fisher Scientific, 1:5000, 31470). The blots were developed using Amersham ECL detection reagent (GE Healthcare) and a ChemiDoc Imaging System (BioRad).

Proteins were separated in SDS-PAGE gels and the separated proteins were transferred to PVDF membranes. The membranes were then blocked using 5% non-fat milk in PBST buffer (1 × PBS + 0.1% Tween 20) (PBSTM) for 30 min at room temperature with 60 r.p.m. shaking. The appropriate antibodies were then added to the PBSTM. Antibodies used were: anti-GST (1:5000; #M20007; Abmart), anti-His (1:5000; #M30111; Abmart), Anti-Flag (1:5000; #F3165; Sigma-Aldrich), anti-GFP (1:5000; #M20004; Abmart), and anti-RFP (1:5000; #5f8; Chromotek). After addition of the antibodies, the membranes were incubated at room temperature for 2–4 hr or 4°C for overnight; then washed three times (5 min each) with PBST buffer. The membranes were then incubated with a goat anti-mouse (Odyssey, no. 926 – 32210; Li-Cor) or anti-rabbit (Odyssey, no. 926–32211; Li-Cor) IRDye 800CW antibody (Odyssey, no. 926 – 32210; Li-Cor) at a dilution of 1:10,000 in PBSTM at room temperature for 30 min with 60 r.p.m. shaking; and followed by three washes (5 min each) with PBST. The signals were detected by excitation at 700 and 800 nm using a double color infrared laser imaging system (Odyssey, LI-COR company).

For co-expression with different constructs, agrobacteria were mixed, infiltrated into leaves and harvested at the indicated time points. For the block of α-mannosidases, 50 μM kifunensine (Santa Cruz Biotechnology) was co-infiltrated with the agrobacteria suspension and for the block of α-glucosidases 200 μM castanospermine (Sigma-Aldrich) was co-infiltrated. Crude protein extracts or purified protein were subjected to SDS-PAGE under reducing or non-reducing (no reducing agent, no boiling of samples) conditions and after blotting the proteins were detected using anti-His (Thermo Fisher Scientific), anti-RBD (Sino Biological) anti-GFP-HRP (Miltenyi Biotec), anti-RFP (Chromotek) and anti-HA (Roche) antibodies. For deglycosylation, proteins were denatured and incubated with or without Endo H or PNGase F (both from NEB) according to the manufacturer’s instructions.