Rabbit anti fasn

Cell lysates derived from Myc-Cap cells and appropriate tissue or cell line controls were separated by SDS/PAGE^{19 (link)} and Western blot performed. Antibodies used were mouse anti-PSMA (clone: OTI2E1; 2 µg/mL; Thermo Fisher), rabbit anti-HPN (1 µg/mL; OriGene), mouse anti-AMACR (clone: OTI5F10; 2 µg/mL; Novus Biologicals) rabbit anti-FASN (diluted 1:1000; Cell Signaling Technology), rabbit anti-PSGR (diluted 1:500; Novus Biologicals), rabbit anti-AGR2 (clone: D9V2F; diluted 1:500; Cell Signaling Technology), rabbit anti-ERG (clone: EPR3864; diluted 1:1000; abcam), rabbit anti-CRISP3 (clone: D-6; diluted 1:100; abcam) and HRP-conjugated goat anti-rabbit and rabbit anti-mouse (diluted 1:10,000; Invitrogen).

Immunoblots were performed as described previously (5 (link)). Briefly, cell pellets was suspended in RIPA lysis buffer (5 (link)). Protein concentration was determined using bicinchoninic acid assay (Pierce). Equal amount of total protein was loaded and separated by SDS-PAGE and transferred onto polyvinylidene fluoride membrane (Millipore). The membranes were blocked with 4% BSA/Tris-buffered saline with 0.1% Tween 20 detergent (TBS-T) and then probed with corresponding antibodies. The following antibodies were used: mouse anti-β-actin (Sigma, #A5316, RRID:AB_476743), rabbit anti-SCD1 (Abcam, #ab236868, RRID: AB_2928123), rabbit anti-CARM1 (Cell Signaling Technology, #3379S, RRID:AB_2068433), rabbit anti-FASN (Cell Signaling Technology, #3189, RRID:AB_2100798), rabbit anti-ACC1 (Cell Signaling Technology, #4190, RRID:AB_10547752), rabbit anti-cleaved PARP (Cell Signaling Technology, #5625S, RRID:AB_10699459), and rabbit anti-Lamin A/C (Cell Signaling Technology, #2032S, RRID:AB_2136278).

For immunofluorescence experiments, 40–50 μm floating sections were collected in 1× PBS, then rinsed once in 1× PBT (PBS + 1% Triton 100×), and incubated overnight at 4 °C in a cocktail of primary antibodies that were diluted in 1× PBT + 20% normal donkey serum. After the overnight incubation, sections were rinsed three times with 1× PBT for 10 min and incubated for 1.5 h in the corresponding secondary antibodies (1:500, Jackson-ImmunoResearch or Invitrogen). Cerebellar sections were then washed three times with 1× PBT for 10–15 min, incubated with DAPI, and mounted in Poly-aquamount (Polysciences). The following primary antibodies were used: rabbit anti-IBA1 (1:200, Wako, # 019-19741), mouse anti-CALB (calbindin, 1:200, Sigma-Aldrich, #C9848), rat anti-CD68 (1:200, Bio-Rad, #MCA1957), Lycopersicon esculentum(Tomato)-Lectin (1:200, Sigma-Aldrich, #L0401), guinea-pig anti-VGLUT1 (1:800, Synaptic Systems, #135404), rabbit anti-phosphorylated S6R (1:200, Cell Signaling, #2211), rabbit anti-phosphorylated AKT (1:200, Cell Signaling, #8112), rabbit anti-TFEB (1:200, Bethyl Laboratories, A303-673A), rabbit anti-FASN (1:200, Cell Signaling, #3180), rabbit anti-pyruvate dehydrogenase E 1 alpha (PDE) (1:200, GeneTex, # GTX104015), rat anti-CD107a (LAMP-1) (1:200, BioLegend, # 121602).