*2.4. Transcript Analysis by qRT-PCR*

Cells were lysed and homogenized using QIAshredders (QIAGEN, Hilden, Germany), and total cellular RNA was isolated using a RNeasy Kit (QIAGEN) with DNase I (QIAGEN) digestion on column. Purified RNA was converted to complimentary DNA (cDNA) using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). The resulting cDNA was analyzed by qRT-PCR using SYBR Green Master Mix (ThermoFisher, Waltham, MA, USA) and gene-specific primers on the ABI 7500 Real-Time PCR System.

## *2.5. Protein Analysis by Western Blot*

Cells were washed 1 x with phosphate bu ffered saline (PBS) and then lysed in radioimmunoprecipitation assay (RIPA) bu ffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 1:100 phosphatase inhibitor cocktail (VWR, Radnor, PA, USA), 1:100 protease inhibitor cocktail (Sigma, St. Louis, MO, USA), 250 nM okadaic acid) on ice. Lysates were scraped into microfuge tubes and immediately frozen at −80 ◦C. Lysates were subsequently thawed on ice for >30 min, then sonicated in a water bath ice slurry for 3 × 30 s bursts on a high setting with 2 × 20 s pauses between. Nuclei and other cellular debris were cleared from lysate via centrifugation at 14,000 rpm for 15 min at 4 ◦C. Protein content was quantified using a Bio-Rad Protein Assay (Bio-Rad), and samples were stored at −80 ◦C until required.

Prior to loading, lysates were incubated with 4X Laemmli bu ffer +/− β-mercaptoethanol at 95 ◦C for 3 min. Samples were loaded (≈7–14 μg total protein per lane) onto 4–20% Criterion TGX gels (Bio-Rad) and electophoresed at 97 V in Western running bu ffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS). Subsequently, proteins were transferred to nitrocellulose membranes at 90 V for 1 h under submerged conditions in Western transfer bu ffer (25 mM Tris, 192 mM glycine, 0.01% (w/v) SDS, 20% (v/v) methanol). Membranes were blocked for 1 h at RT in Tris-bu ffered saline (TBS)-based Odyssey blocking bu ffer (LI-COR, Lincoln, NE, USA), and subsequently stained overnight with primary antibody (see key resources table) in TBS-based Licor blocking bu ffer at 4 ◦C. The following day, membranes were

washed 3x with TBS plus Tween 20 (TBST), and probed with secondary antibody (either horseradish peroxidase (HRP)-, Alexa680-, or Alexa790-conjugated antibody; key resources table) in TBS-based Odyssey blocking bu ffer for 1 h at RT. Finally, membranes were washed 3x with TBST and 2x with TBS prior to either direct imaging on an Odyssey CLx imager (LI-COR), or band development using enhanced chemiluminescence (ECL) prime Western blotting reagen<sup>t</sup> (Fisher Scientific) and detection on a ChemiDoc XRS+ system (Bio-Rad).
