Ha antibody

NT‐PGC‐1α‐HA and Flag‐HNF4α were co‐transfected into HEK293 cells using Fugene 6 (Roche, Indianapolis, IN). Immunoprecipitation assays were carried out as described previously by Chang et al. (2010). Briefly, whole cell extracts were incubated with Flag antibody (Sigma) or HA antibody (Abcam, Cambridge, MA) overnight at 4°C, followed by incubation with protein G‐agarose beads. The immunoprecipitates were extensively washed, separated by SDS‐PAGE, and immunoblotted with HA or Flag antibody.

After treatment at the indicated time, cells were harvested, and the proteins from cells were separated by SDS–PAGE in 12% (w/v) polyacrylamide gels and transferred to PVDF membranes. The membranes were then incubated with the appropriate primary antibody overnight at 4 °C. After subsequently rinsing in PBS, membranes were incubated with secondary antibody conjugated to horseradish peroxidase, the bands were visualized by enhanced chemiluminescence (Thermo Fisher Scientific, San Jose, CA, USA), and the blots were exposed to film (Thermo Fisher Scientific, San Jose, CA, USA) [28 (link)]. Flag antibody (Sigma, St. Louis, MO, USA), HA antibody (Abcam, Cambridge, MA, USA), PKM2 antibody (Cell Signaling Technology, Danvers, MA, USA), HNF4α antibody (Santa Cruz, CA, USA) were used for protein assay.

Antibodies to c-Myc, Mcl-1, survivin, Bcl-2, eIF4E, eIF4G and RLP26 were from Cell Signaling Technology (Beverly, MA, USA). HA antibody was from abcam (Cambridge, MA USA). Actin antibody was purchased from Santa Cruz (Santa Cruz, CA, USA).

To analyze the expression level of HSI-I, HA-tagged TssB1 was knocked into the genome of P. aeruginosa replacing the original TssB1. In order to standardize the TssB1-HA expression level, RNA polymerase II (RNAP) was selected as an internal reference for western blot analysis. Strains were cultured overnight in 3 ml TSB with shaking in a 37 °C incubator, then a 1:100 dilution was grown in 20 ml TSB at 37 °C to OD₆₀₀ of 1.0. Cells were harvested by centrifugation (5000 rpm for 10 min, 4 °C), and ultrasonic lysed with 15 ml ice pre-cold cell lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF pH 8.0) for 15 min until cells were completely lysed. 300 μl cell lysates were collected and mixed with 100 μl the 4× SDS-PAGE sample loading buffer. The mixtures were boiled at 100 °C for 10 min, subjected to SDS-PAGE gel, and electroblotted onto a polyvinylidene difluoride membrane (Amersham BiosciencesUK). The membranes were blotted with the HA antibody (Abcam) (1:2000 dilutions) or RNAP antibody (Abcam) (1:2000 dilutions), followed by the Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (Proteintech) (1:50,000 dilutions). Signals were detected with the Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA), and images were captured with the Analytik-jena imaging system. Band intensities were quantified using the ImageJ software (NIH, Bethesda, MD, USA).

Six NAc tissue punches (2 mm) from each RT; D1-Cre or RT; D2-Cre mouse are homogenized in 1 ml of homogenization buffer [8 (link)] for each sample (two mice, which have similar SI ratio, pooled for each sample). Samples are then centrifuged at 4 °C, 10,000 g for 10 min and 800 μl of clear supernatants are mixed with 5 μg of HA-antibody (Abcam, Cambridge, MA), and incubated on a gentle rotator at 4 °C overnight. The next day, 200 μl of magnetic beads (Dynabeads anti-rabbit IgG; Life technology, Carlsbad, CA) are added and incubated overnight with constant rotation. The following day, magnetic beads are washed three times in the magnetic rack for 10 min with a high salt wash buffer. Each RNA sample was extracted using the Direct-zol miniprep kit (Zymo Research, Irvine, CA) and subjected to qRT-PCR or RNA-seq. For quality control, RNA integrity (RIN) values were measured and samples with RIN < 7 were excluded.