Pka riiβ

Arl13b, AKAP9, Gli2, Gli3, and pGli antibodies were generated by Covance or Biosynthesis. Rabbits were immunized with purified bacterially expressed His-tagged mouse Arl13b, His-tagged mouse AKAP9 fragment (2768–3116 aa), His-tagged mouse Gli2C-terminal fragment (605–1460 aa), GST-tagged Gli3 C-terminal fragment (1051–1179 aa), or the synthetic Gli2P2 phospho-peptide (NH₂-CAYTVSRRS-(pS)-GISP-OH), in which residue C is not from Gli2 and was used for conjugation of the peptide to Keyhole Limpet Hemocyanin (KLH). It is worth noting that the sequence for Gli3 in the same region is AYLSSRRSSGISP with only the 3rd and 4th residues different from the Gli2. The phosphorylation site is conserved between Gli2 and Gli3. The antibodies were used in a 1:1000 dilution. Other antibodies include: Gli2 N-terminal, Gli3 N-terminal, Ta3, Cep120 (all 1:1000) (Pan et al., 2006 (link); Wang et al., 2000 (link); Wu et al., 2014 (link)), acetylated tubulin (1:4000), FLAG (T6793, F1804, Sigma), PKARIIβ (610625, BD Biosciences), and Myc (sc-788, Santa Cruz Biotechnology). Secondary antibodies Alexa Fluor 488-conjugated goat anti-rabbit IgG (111−545-144) and Cy3-conjugated goat anti-mouse IgG (115−165-146) were purchased from Jackson Immunoresearch, Inc.

Human FL-HCCs and normal liver samples were homogenized in ice-cold radioimmunoprecipitation buffer containing protease inhibitors, and protein concentrations were measured using the BCA Protein Assay (Pierce, Rockford, IL). Equal amounts of protein were separated by sodium dodecyl sulfate – poly-acrylamide gel electrophoresis (SDS-PAGE), transferred to Immobilin-P membranes (Millipore, Bedford, MA) and incubated at 4 °C overnight with the following primary antibodies: PKA C (SC-903, Santa Cruz Biotechnologies, Santa Cruz, CA), PKA RIIα (SC-909, Santa Cruz Biotechnologies), PKA RIIβ (610626, BD Biosciences, San Jose, CA), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), total p44/42 MAPK (Erk1/2), and PKA RIα (9101, 9102, 5675, Cell Signaling, Danvers, MA), and β-Actin (A5441, Sigma, St. Louis, MO). Densitometry analysis was performed using NIH ImageJ software (National Institutes of Health, Bethesda, MD) by measuring the integrated density around each band and normalizing to the integrated density of the actin band for each sample.