Validation of Tankyrase Binding and PARylation in Full-Length Protein Context

Grow a sufficient number (here two) 15 cm cell culture plates of HEK293T cells to a cell density of ≈90% confluence in standard DMEM media supplemented with 10% FBS.

Remove the media and gently rinse the cell culture plates with 10mL to remove residual media, which could impede the efficient dissociation of the cells by trypsin.

Add 3mL to each plate and place the plates back in the incubator for 0h 2m 0s. Gently tap the side of the plates to help the cells detach from the plate and dissociate from each other.

Add 10mL to each plate; the FBS will inactivate the trypsin. Pipette the cell suspension up and down a few times to ensure proper dissociation of cell clumps into a homogenous cell suspension.

Measure the cell density, using either a hemocytometer or automated cell counter. Add 6 × 10⁶ cells each to the required number of 10 cm cell culture plates for the co-transfection of different DNA constructs (Table 3). Add 10mL to each plate and incubate cells 0h 2m 0s.

A	B	C	D	E	F	G	H
	empty FLAG vector	MERIT40 WT	MERIT40 G33R	MERIT40 G53R	MERIT40 GG33/53RR	TRF1 WT	TRF1 G18R
empty MYC2 vector	−	+	−	−	−	+	−
TNKS2 WT	++	+	+	+	+	+	+
TNKS2 G1032W	−	+	−	−	−	+	−

Table 3 Chart for co-transfection of HEK293T cells for co-immunoprecipitation. The indicated FLAG/FLAG3-tagged constructs (5 μg) are used as baits in co-immunoprecipitation with the indicated MYC2-tagged TNKS2 constructs (5 μg). The G-to-R mutation at position 6 of the TBM efficiently abolishes the ARC–TBM peptide interaction [7]. A G1032W mutation abolishes both poly- and mono(ADP-ribosyl)ation by TNKS2 [31]. Each "+" sign denotes a single co-transfection setup

The next day, co-transfect cells with 5 μg of each DNA construct (thus 10 μg total) per plate (Table 3):

Pipette 5µg in a 15 mL Falcon tube, add 250µL followed by 1.75mL and mix thoroughly. Subsequently, add 2mL, mix the solution thoroughly by pipetting up and down and incubate for 0h 5m 0s–0h 10m 0s at Room temperature.

Replace the growth media from the 10 cm plate with 15mL supplemented with 25micromolar (µM). Chloroquine inhibits degradation of endocytosed DNA and can increase transfection efficiency [21].

Gently add the transfection mix (4 mL each, dropwise) to each plate and incubate transfected cells for 24h 0m 0s.

The next day, remove the media and add 10mL. Harvest the cells by gently scraping them off the plate.

Collect the scraped cell suspension in a 15 mL Falcon tube and pellet the cells by centrifugation at 300x g,4°C. Discard the supernatant; snap-freeze cell pellet in liquid nitrogen and store at -80°C until further use.

Add 1mL (with freshly added DTT and protease inhibitors) to each cell pellet and resuspend cells On ice. The freeze–thaw and the detergents will break open the cells. Transfer the lysates to 1.5 mL microcentrifuge tubes and incubate On ice for 0h 5m 0s for extraction.

Sonicate lysates for 0h 0m 6s On ice, using a small tip sonicator at 25% amplitude output to shear chromatin.

Clear cell lysates by high-speed centrifugation at 18000x g,4°C and transfer supernatants to new 1.5 mL microcentrifuge tubes.

Transfer 30µL to a new microcentrifuge tube ( input samples ) and add 10µL. Boil samples at 95°C for 0h 5m 0s, collect by brief centrifugation , and store at -20°C until analysis, if required.

Pre-equilibrate a sufficient amount of anti-FLAG M2 Agarose resin (25µL per immunoprecipitation sample plus a minimum of 10% dead volume) with RIPA buffer and incubate resin with cleared lysates for 2h 0m 0s on a rotating wheel at 4°C.

Gently settle the resin by centrifugation at 800x g,4°C and remove supernatant using a vacuum pump.

Wash the resin by adding 1mL to each sample and incubating microcentrifuge tubes for 0h 5m 0s on a rotating wheel at 4°C.

Settle the resin as before (step 16: Gently settle the resin by centrifugation at 800x g,4°C and remove supernatant using a vacuum pump.) and repeat the wash step four more times:

Gently settle the resin by centrifugation at 800x g,4°C and remove supernatant using a vacuum pump. (Wash 1/4)

Gently settle the resin by centrifugation at 800x g,4°C and remove supernatant using a vacuum pump. (Wash 2/4)

Gently settle the resin by centrifugation at 800x g,4°C and remove supernatant using a vacuum pump. (Wash 3/4)

Gently settle the resin by centrifugation at 800x g,4°C and remove supernatant using a vacuum pump. (Wash 4/4)

After the last wash step, gently remove as much of buffer as possible and add 25µL. Boil samples at 95°C for 0h 5m 0s, collect briefly by centrifugation and store at -20°C until analysis, if required (immunoprecipitate (IP) samples).

Resolve 10µL (step 14 above) and IP (step 19 above) samples on a pre-cast 4–15% Tris–glycine polyacrylamide gradient gel for SDS-PAGE analysis.

Transfer proteins from the gel onto a nitrocellulose membrane using a wet-transfer blotting system. Ponceau S can be used to assess the transfer quality.

Incubate the nitrocellulose membrane for at least 1h 0m 0s at Room temperature on a horizontal shaking platform in 5% dry milk powder in PBS to block the membrane to reduce nonspecific binding of antibodies during the subsequent immunodetection steps.

Incubate the membranes with the required antibody at the appropriate dilution in 5% (v/v) 1h 0m 0s at 4°C on a horizontal shaking platform. (Antibody dilutions: anti-FLAG HRP-conjugated antibody, 1:1000; anti-MYC HRP -conjugated antibody, 1:1000; anti-PAR, 1:1000) .

Wash the membrane three times for 0h 5m 0s with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform:

Wash the membrane for 0h 5m 0s with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform. (Wash 1/3)

Wash the membrane for 0h 5m 0s with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform. (Wash 2/3)

Wash the membrane for 0h 5m 0s with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform. (Wash 3/3)

As required, incubate the membrane with the matching HRP-coupled secondary antibody, here diluted 1:5000 in 5% (v/v) for 1h 0m 0s at Room temperature on a horizontal shaking platform ( see Note 20 ).

Repeat wash: Wash the membrane three times for 0h 5m 0s with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform:

Wash the membrane for 0h 5m 0s with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform. (Wash 1/3)

Wash the membrane for 0h 5m 0s with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform. (Wash 2/3)

Wash the membrane for 0h 5m 0s with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform. (Wash 3/3)

Develop Western blot by incubating membrane with ECL Western blotting substrate following the manufacturer’s instructions.

Detect chemiluminescence signal by exposing membrane to X-ray film in a dark room and develop the film with an X-ray film developer.

Fig. 4 shows the TBM-dependent interaction of FLAG3-TRF1 and FLAG-MERIT40 with MYC2-TNKS2, and the TNKS2-dependent PARylation of both TNKS2 interactors, in addition to TNKS2 auto-PARylation.

Both MERIT40 TBMs contribute to tankyrase binding (Fig. 4b).