Ripa buffer

Efficacy for CRISPR/Cas9-mediated ablation of AIM2, caspase-1, STING, cGAS and IFI16 proteins in human THP-1 cells was evaluated by western blot. For AIM2 detection, control or AIM2 KO THP-1 cells were treated with human IFN-γ (75ng/ml, R&D System) or infected with Ad5 or ALVAC vector for 24 hours, followed by cell lysis in RIPA buffer (Cell Signaling). For detection of caspase-1, STING, cGAS and IFI16, control and KO THP-1 cells are directly lysed in RIPA buffer (Cell Signaling). Cell lysates were separated by 10% SDS-PAGE and transferred to PVDF membranes (Bio-rad). After blocking in 5% non-fat milk (TBS+0.1% Tween 20), membranes were incubated with anti-AIM2 antibody (1:500, Cell Signaling), anti-caspase-1 antibody (1:1000, Thermo Scientific), anti-STING antibody (1:1000, Cell Signaling), anti-cGAS (1:1000, Cell signaling), or anti-IFI16 (1:1000, Thermo Scientific) at 4°C overnight and developed with Rabbit IgG Horseradish Peroxidase-conjugated Antibody (1:1000, Cell Signaling) or mouse IgG Horseradish Peroxidase-conjugated Antibody (1:1000, Cell Signaling) and SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific). β-actin was also detected as control, using anti-β-actin antibody (1: 50,000, R&D System) for incubation at room temperature for 1 hour and then mouse IgG Horseradish Peroxidase-conjugated Antibody (1:3000, Cell Signaling) after striping.

To measure MYOD mRNA half-life at day 0 (following transfection of 54-1 cells with siControl #1 or siUPF1 #1), actinomycin D (ActD, Sigma, 2.5μg/mL) was added to cells to inhibit transcription. After 0, 0.5, 1, 2, 4, 8 and12 hours of ActD treatment, cells were collected with TRIzol (Invitrogen), RNA was extracted, and qPCR was carried out with primers specific to mature MYOD mRNA. To inhibit the 26S proteasome, MG132 (10μM) was added to MB135-Tet-UPF1^WT cells 12 hours post-Dox induction. After 8 hours of MG132 treatment, identical numbers of cells were collected in RIPA buffer (Cell Signaling) supplemented with protease inhibitor tablets (Fisher Scientific), protein was extracted, and immunoblots for MYOD were performed. To measure MYOD protein levels in MB135-Tet-UPF1^WT or MB135-Tet-UPF1^{S124A/N138A/T139A} cells that were or were not treated with Dox to induce transgenic UPF1 expression, cycloheximide (CHX, 100μg/mL) was added to cells 12 hours post-Dox induction to inhibit translation. After 0, 2, 4, 6 and 8 hours of CHX treatment, identical numbers of cells were collected in RIPA buffer (Cell Signaling) supplemented with protease inhibitor tablets (Fisher Scientific), protein was extracted, and immunoblots for MYOD were performed.

Cells were harvested, washed with PBS and lysed in 1X RIPA buffer (Cell Signaling, USA) containing protease and phosphatase inhibitor cocktails with constant rotation at 4 °C for 30 min. Cell lysates (2000–2200 μg) were incubated with RARβ, ING4, or rabbit IgG control antibodies together with protein G Sepharose beads (GE healthcare, USA) at 4 °C overnight. The immune complexes were isolated via centrifugation and washed four times with 1X RIPA buffer. The proteins were then separated with an SDS/polyacrylamide gel.

Isolated synaptosomes or P0–P2 FVB or FX DIV14–16 cortical cultures were treated for 15 min. with puromycin. 50–100 μg of protein from cell lysate, was incubated at 4°C overnight with 1μg of anti-puromycin antibody (Kerafast). Next, 50 μl of protein G agarose (Roche Diagnostics GmbH) was added to samples for an overnight incubation at 4°C. Beads were washed 3 times with 1x RIPA buffer (Cell Signaling) and processed for mass spectrometry analysis. For experiments with Dexpramipexole, cortical cultures were treated with 10μM Dex for 2–24 hours prior to puromycin treatment and then processed for western blot.