Passive lysis buffer (plb)

Cells were harvested 48 h after transfection
(or for kinetic experiments at indicated time points) and lysed using
25 μL of Passive lysis buffer (Biotium). Firefly and Renilla
luciferase activity was measured in cell lysate using the dual luciferase
assay (Promega), and luminescence was measured on a Centro microplate
luminometer (Berthold Technologies). Relative luciferase units (RLU)
were calculated by normalizing each sample’s luciferase signal
to the renilla signal of the same sample.

Forty-eight hours after transfection,
cells were washed with PBS and resuspended in 200 μL of Passive
lysis buffer (Biotium) and lysed for 30 min on ice. After, the lysates
were centrifuged for 5 min at 14,200 rpm on a tabletop centrifuge
to remove cell debris. The total protein concentration was measured
in the supernatant using a BCA assay (Sigma-Aldrich). Then, 30 μg
of each sample was denatured by incubating the sample at 95 °C
for 5 min with SDS. Samples were loaded on 10% SDS-PAGE gels and separated
at 200 V for 40 min. Proteins were transferred to a nitrocellulose
membrane using an iBlot 2 gel transfer device (Invitrogen) according
to the manufacturer’s protocol. Membrane blocking, washing,
and antibody binding were performed using the iBind Flex Western device
(Invitrogen) according to the manufacturer’s protocol. Primary
antibodies were rabbit Anti-HA (Merck, diluted 1:1000) and mouse β-actin
(Cell Signaling Technology, diluted 1:5000). Secondary antibodies
used were goat antirabbit (Abcam, diluted 1:4000) and goat antimouse
(Jackson ImmunoResearch, diluted 1:3000). A signal was developed with
a SuperSignal West Pico (Thermo Fischer Scientific) substrate according
to manufacturer’s protocol, and blots were visualized on a
G-box device (Syngene).

The sequence of the putative PRINS binding site of IL-23 cDNA that was identified in silico was synthesized and inserted into the pmirGLO vector (Promega, Madison, WI, USA) between the Dra I and Xba I restriction sites, resulting in the pmirGLO-IL23BS construct (Supplementary Figure S4). HEK293 cells were transiently transfected by the PRINSpcDNA3.1 or pmirGLO plasmid as controls. Cells were also co-transfected with 0.5 μg pmirGLO-IL23BS or empty pmirGLO plasmid together with the PRINSpcDNA3.1 vector. In each transfection experiment, 0.025 μg renilla-luciferase-expressing pGL4.75 hRluc/CMV (Promega) plasmid was used as internal control. Cells were harvested after 24 h, washed with PBS (phosphate-buffered saline) and lysed with passive lysis buffer (Biotium, Hayward, CA, USA), and luciferase activities were measured using the Firefly and Renilla Dual Luciferase Assay Kit (Biotium) and SYNERGY/HTX Multi-Mode reader (Bio Tek Instruments, Winooski, VT, USA), according to the manufacturer’s instructions. The luciferase activity derived from the pmirGLO plasmids was normalized to the renilla-luciferase activity.

Luciferase assays were conducted as previously described [26 (link)]. Cells were seeded in quadruplicate in a 24-well plate (5 × 10⁴ cells per well) and transfected with Lipofectamine 2000. Each reaction contained 100 ng of the luciferase reporter plasmid and 2 ng pLRL-SV40 Renilla, serving as a transfection control. Total concentration of DNA was adjusted to 500 ng per reaction using pBluescript II SK (-). Where indicated, 50 ng of pcDNA3.1-β-catenin S45F and 50 ng of pME18-Lef1 were added to the transfection reaction. Transfection media were replaced after 6 h. For Dox-induced TCF7L1 or active Wnt signaling experiments, transfection media were replaced with Dox-treated media and/or Wnt3a-conditioned media (L-Wnt3a, ATCC, CRL-2647, Gathersburg, MD, USA), respectively. After 24 h, cells were lysed in passive lysis buffer (Biotium, #99821, Fremont, CA, USA) and luciferase levels were measured using the dual luciferase single tube assay kit (Biotium, #30081) on a Glomax 20/20 single chamber luminometer (Promega, Madison, WI, USA).