Ab27671

Cells were prepared for immunofluorescence microscopy according to reference 14. The V5 epitope was detected with mouse anti-V5 monoclonal antibody SV5-Pk1 (1:750; AB27671; Abcam). The primary antibody was detected with an Alexa Fluor 488-conjugated goat anti-mouse polyclonal antibody (1:800; A11029; Life Technologies). Tubulin was labeled with a mouse anti-tubulin TAT1 monoclonal antibody (1:150) (41 (link)). The primary antibodies were detected with an Alexa Fluor 488-conjugated goat anti-mouse polyclonal antibody (1:250; A-11001; Life Technologies) or an Alexa Fluor 594-conjugated goat anti-rabbit antibody (1:250; catalog no. A-11037; Life Technologies). The cells were viewed with a Zeiss Axioplan 2 fluorescence microscope or a Zeiss 510 laser scanning confocal microscope. The images were processed with the Zen 2011 v7.0.0.285 software (Carl Zeiss GmbH) or the BioImageXD v1.0 RC3 software (42 (link)).

Coverslips were coated with ConA, then washed with PBS before adding 20 µL of parasite culture in PBS. Parasites were allowed to settle for 15 min before washing gently with PBS 3×. Parasites were fixed in 4% PFA and 0.0075% GA for 30 min, ensuring the coverslips were not allowed to dry. Parasites were washed then permeabilized in 0.1% Tx-100 for 10 min, then washed with PBS and blocked for 30 min with 1% BSA in PBS. The primary antibody was added for 1 hr, before washing 3× with PBS, and adding the secondary for 1 hr. After five washes with PBS, a drop of ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) was added to each coverslip before mounting onto a slide for imaging.
For detecting FIKK4.1-HA, cells were probed with rat anti-HA (clone 3F10, Roche; 1:1000), followed by 1:10,000 anti-rat Alexa 594 secondary. For detecting biotinylated proteins, cells were probed with 1:5000 streptavidin-Alexa 488. For detecting KAHRP or PTP4 V5, cells were probed with either rabbit anti-V5 (MA5-32053, Invitrogen; 1:200), followed by 1:10,000 anti-rabbit Alexa 488, or with mouse anti-V5 (ab27671, Abcam, 1:200) followed by 1:10,000 anti-mouse Alexa 488 (all secondary antibodies from Life Technologies).
Parasites were imaged with a VisiTech iSIM microscope, and images were deconvolved and analyzed in Huygens Professional software.

Protein extracts in sample buffer were separated on Bolt 4%-to-12% bis-Tris Plus gels (Life Technologies, Inc.) and transferred to Protran 0.45-μm nitrocellulose membranes (GE Healthcare Life Sciences) by using the Trans-blot SD semidry transfer cell (Bio-Rad). Membranes were blocked for a minimum of 1 h in 5% (wt/vol) nonfat dry milk in Tween-PBS (PBS with 0.1% Tween 20). Following this, the membrane was incubated for 16 h at 4°C in 2% (wt/vol) nonfat dry milk in Tween-PBS containing either mouse anti-myc (1:1,000 dilution; catalog number 2276; Cell Signaling), mouse anti-V5 (1:2,000, AB27671; Abcam, Inc.), or mouse anti-tubulin (1:5,000; T5168; Sigma-Aldrich). Thereafter, membranes were washed three times for 10 min with Tween-PBS and incubated for an hour in 2% (wt/vol) nonfat dry milk in Tween-PBS with anti-mouse secondary antibody conjugated with horseradish peroxidase (A16072; Life Technologies, Inc.). This was followed by three 10-min washes with Tween-PBS buffer, and enhanced chemiluminescence was used for signal detection (Thermo Fisher Scientific).

1.5 × 10⁶ of 293T cells infected with GIPZ-shTMED3 or control lentiviruses were seeded in 6-cm dishes in DMEM with 10% FBS. The following day the infected cells were transfected using calcium phosphate with 0.8 μg of V5-tagged WNT3A expression plasmid (Addgene 35927). Medium was replaced 6 h after by 4 ml of DMEM with 0.5% FBS. Supernatants were collected 48 h later, spun twice on a table top centrifuge to remove any floating cells or debris and concentrated ∼20-fold (Pierce Concentrators 9K MWCO, 89884A, Thermo Scientific). Cells were also collected directly from the dish and lysed in RIPA buffer. 25 μg of total protein from the concentrated supernatants or 2 μg from the cell lysates were loaded per well onto 12% SDS–PAGE. Western blotting was performed using anti-V5 (ab27671, Abcam, 1/500 overnight) and anti-PCNA (SC-7907, Santa Cruz, 1/1,000 1 h) antibodies.

For co-immunoprecipitation, cells were lysed in 1% NP-40 in TBS plus 10 mM iodoacetamide, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and benzonase (Sigma-Aldrich) for 30 min. Protein A and IgG-sepharose resin was added to the lysates along with primary antibody. The suspension was incubated for 2 h at 4 °C and the resin was washed three times in lysis buffer. For western blotting, cells were lysed with lysis buffer containing 1% SDS instead of 1% NP-40. For SDS-PAGE analysis, resins or lysates were heated to 70˚C in SDS sample buffer for 10 min and run on a polyacrylamide gel. Gels were blotted onto PVDF membranes (Millipore). Blots were blocked in 5% milk in PBS, 0.2% Tween-20 and incubated with primary antibody diluted in blocking solution. As the Periphilin antibody was unable to detect its epitope under NP-40 lysis conditions, we used a mouse antibody against the V5 tag (Abcam, ab27671) as the primary antibody for Periphilin. For TASOR, the primary antibody was rabbit α-TASOR (Atlas, HPA006735). Blots were imaged with West Pico or West Dura (Thermo Fisher Scientific).

Detecting reagents, including streptavidin-HRP (1:2,000 for WB, 3999S), anti-Myc antibody (1:2,000 for WB, 2276S), anti-Myc HRP (1:1,000 for WB, 2040S), anti-HA antibody (1:2,000 for WB, 2367S), anti-HA HRP (1:1,000 for WB, 2999S), V5 (1:1,000 for immunofluorescence, 13202S), and anti-GFP HRP (1:1,000 for WB, 2037S) were purchased from Cell Signaling Technology. Antibodies against V5 (1:3,000 for WB, ab27671) and MARCH5 (1:2,000 for WB, ab174959) were purchased from Abcam. Anti-GDAPH (1:4,000 for WB, A00191) and anti–β-actin (1:4,000 for WB, A00702) were purchased from GenScript. Other antibodies used in this study included anti-FLAG (1:2,000 for WB, GNI14110-FG) and anti-FLAG HRP (GNI4310-FG; GNI), anti-PMP70 (1:3,000 for WB, SAB4200181; Sigma-Aldrich), and anti-GFP (1:3,000 for WB, M20004; Abmart). Anti–V5-HRP (1:5,000 for WB, R961-25) was purchased from Thermo Fisher Scientific. Other primary antibodies used were anti-MARCH5 (19168S; Cell Signaling Technology), anti-PEX19 (14713–1-AP; Proteintech), anti-PEX3 (sc-271477; Santa Cruz Biotechnology), anti-catalase (219010; Millipore), and anti-PEX13 (ab235043; Abcam). Secondary antibodies conjugated to HRP were purchased from GenScript.