Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Hepatic adenomas with synchronous or metachronous fibrolamellar carcinomas: both are characterized by LFABP loss.

doi: 10.1038/modpathol.2016.59

Figure Lengend Snippet: Figure 4 PRKAR1A expression in fibrolamellar carcinomas. (a) The fibrolamellar carcinoma negative for the DNAJB1- PRKACA fusion shows loss of PRKAR1A expression. (b) In contrast, fibrolamellar carcinomas with the DNAJB1-PRKACA fusion show retained expression of PRKAR1A.

Article Snippet: Five micron-thick sections were cut from the tissue block and immunohistochemistry performed using antibodies to Arginase-1(predilute, clone SP156; Cell Marque), HepPar1 (predilute, clone OCH1E5; Ventana), cytokeratin 7 (1:100, Clone OV-TL 12/30; Dako), CD68 (clone KP1, 1:50-1:100; Dako), PRKAR1A (1:2000, clone OTI6C7; Origene Technologies), LFABP (1:50, rabbit polyclonal; Abcam), CRP (1:2000, clone Y284; Abcam), SAA (1:2000, clone mc1; Dako), Glutamine synthetase (1:2000, clone GS-6; Millipore), and beta catenin (predilute, clone 14; Ventana).

Techniques: Expressing

Journal: Endocrine-Related Cancer

Article Title: Defects of the Carney complex gene (PRKAR1A) in odontogenic tumors

doi: 10.1530/erc-15-0094

Figure Lengend Snippet: Figure 1 Ideogram of the cytogenetic bands of chromosome 17 and polymorphic microsatellite markers and representative results of PRKAR1A loss of heterozygosity (LOH). (A) Polymorphic DNA markers located on the long arm of chromosome 17, including D17S1882 (17q24.1), D17S942 (17q24.2), D17S940 (17q24.2), and D17S2182 (17q24.3), which are used to assess LOH at the PRKAR1A genetic locus. (B and C) Loss of the long allele at marker D17S1882 in the mesenchyme of case 3 (B) and in the tumor of case 5 (C) as compared with matched normal DNA. *short allele, **long allele. A full colour version of this figure is available at http://dx.doi.org/10.1530/ ERC-15-0094.

Article Snippet: Immunohistochemical reactions Published by Bioscientifica Ltd. E n d o cr in e -R e la te d C a n ce r were performed using a mouse MAB against the PRKAR1A protein (clone 6C7) (OriGene, Rockville, MD, USA).

Techniques: Marker

Journal: PLoS Genetics

Article Title: AKAP13 couples GPCR signaling to mTORC1 inhibition

doi: 10.1371/journal.pgen.1009832

Figure Lengend Snippet: (A) AKAP13 interacts with mTORC1. Empty vector (Flag) or Flag-tagged AKAP13 was co-expressed with HA-tagged Raptor in HEK293A cells. 24 hours later Flag immunoprecipitates (IPs) were analyzed by immunoblotting for Flag-tagged AKAP13, HA-tagged Raptor, mTOR, and mLST8. Actin was used as a loading control. WCL denotes whole cell lysate. (B) AKAP13 interacts with PKA Catα and PKA RIIα. Empty vector (Flag) or Flag-tagged AKAP13 was co-expressed with HA-tagged Raptor in HEK293A cells. 24 hours later cells were then treated with or without 10 μM forskolin and 200 μM IBMX for 1 h, and Flag immunoprecipitates (IPs) were analyzed by immunoblotting for Flag-tagged AKAP13, PKA Catα, PKA RIα, and PKA RIIα. CREB phosphorylation (pCREB) at Ser 133 was used as a positive control for forskolin stimulation. Actin and CREB were used as loading controls. WCL denotes whole cell lysate. (C) Left- Elevated AKAP13 levels decrease mTORC1 activity. Flag-tagged AKAP13 (0–3 μg) was overexpressed in HEK293A cells for 24 hours. mTORC1 activity was analyzed by protein immunoblotting for the phosphorylation status of S6K1 (pS6K1) at Thr 389, 4EBP1 (p4EBP1) at Thr 37 and Thr 46, and ULK1 (pULK1) at Ser 758. S6K, 4EBP1, ULK1, and Actin were probed as loading controls. Right - Quantification of phospho-ULK1. P-Values: 0 μg FLAG-tagged AKAP13 vs 0.5 μg FLAG-tagged AKAP13 p<0.05, 0 μg FLAG-tagged AKAP13 vs 1 μg FLAG-tagged AKAP13 p<0.01, 0 μg FLAG-tagged AKAP13 vs 2 μg FLAG-tagged AKAP13 p<0.0001 and 0 μg FLAG-tagged AKAP13 vs 3 μg FLAG-tagged AKAP13 p<0.001. Quantification of phospho-S6K. P-values: 0 μg FLAG-tagged AKAP13 vs 0.5 μg FLAG-tagged AKAP13 p = 0.05, 0 μg FLAG-tagged AKAP13 vs 1 μg FLAG-tagged AKAP13 p<0.01, 0 μg FLAG-tagged AKAP13 vs 2 μg FLAG-tagged AKAP13 p< 0.01 and 0 μg FLAG-tagged AKAP13 vs 3 μg FLAG-tagged AKAP13 p<0.0001. Quantification of phospho-4EBP1. P-values: 0 μg FLAG-tagged AKAP13 vs 0.5 μg FLAG-tagged AKAP13 p<0.05, 0 μg FLAG-tagged AKAP13 vs 1 μg FLAG-tagged AKAP13 p<0.01, 0 μg FLAG-tagged AKAP13 vs 2 μg FLAG-tagged AKAP13 p<0.001 and 0 μg FLAG-tagged AKAP13 vs 3 μg FLAG-tagged AKAP13 p<0.001. (D) Decreased AKAP13 levels increase mTORC1 activity. HEK293A stable cell lines expressing control shRNA (shGFP) or four different shRNA targeting AKAP13 (shAKAP13 1–4) were generated. mTORC1 activity and loading controls were analyzed as described in (C). (E) Left- Elevated AKAP13 levels increase Raptor Ser 791 phosphorylation. HA-tagged Raptor was co-expressed with empty vector (Flag), Flag-tagged AKAP13, or Flag-tagged AKAP8L in HEK293A cells. 24 hours later cells were then treated with or without 10 μM forskolin and 200 μM IBMX for 1 hour, and HA immunoprecipitates (IPs) were analyzed by immunoblotting for HA-tagged Raptor, and phospho-PKA substrate antibody (pPKASub (RRXS*/T*)). CREB phosphorylation (pCREB) at Ser 133 was used as a positive control for forskolin stimulation. Actin and CREB were used as loading controls. WCL denotes whole cell lysate. Right- Quantification of phospho-PKA substrate (pPKASub (RRXS*/T*) to determine Raptor Ser 791 phosphorylation. P-values: Flag -Forskolin vs Flag +Forskolin p<0.0001, Flag-tagged AKAP13 -Forskolin vs Flag-tagged AKAP13 +Forskolin p<0.01, Flag-tagged AKAP8L -Forskolin vs Flag-tagged AKAP8L +Forskolin p<0.01, Flag +Forskolin vs Flag-tagged AKAP8L -Forskolin p<0.0001, Flag +Forskolin vs Flag-tagged AKAP13 +Forskolin p<0.05. (F) Decreased AKAP13 levels decrease Raptor Ser 791 phosphorylation. HEK293A stable cell lines expressing control shRNA (shGFP) or shRNA targeting AKAP13 (shAKAP13) were generated. HEK2943A cells were then treated with or without 10 μM forskolin and 200 μM IBMX for 1 hour, and Raptor immunoprecipitates (IPs) were analyzed by immunoblotting for Raptor and phospho-PKA substrate antibody (pPKASub (RRXS*/T*)). CREB phosphorylation (pCREB) at Ser 133 was used as a positive control for forskolin stimulation. Actin and CREB were used as loading controls. WCL denotes whole cell lysate. (G) Elevated AKAP13 inhibits mTORC1 though Raptor Ser 791 phosphorylation. Flag-tagged AKAP13 was overexpressed for 24 hours in HEK293A or HEK293A Raptor Ser 791 mutant cells (S791A-1, S791A-2). mTORC1 activity and loading controls were analyzed as described in (C).

Article Snippet: AKAP13 (#A301-404A, 1:500), PKA RIα (#A303-683A, 1:1000), PKA RIIα (#A301-670A, 1:1000) was from Bethyl Laboratories.

Techniques: Plasmid Preparation, Western Blot, Control, Phospho-proteomics, Positive Control, Activity Assay, Stable Transfection, Expressing, shRNA, Generated, Mutagenesis

Journal: Nature Communications

Article Title: Capture of RNA-binding proteins across mouse tissues using HARD-AP

doi: 10.1038/s41467-024-52765-w

Figure Lengend Snippet: a Volcano plot showing distributions of proteins captured by HARD beads compared to EGFP beads in indicated samples. The fold changes were calculated from means of the ion intensities of three independent biological samples. The significance (p) was determined using the two-tailed Student’s t-test and further adjusted using the Benjamini-Hochberg correction for multiple testing (p-adjust). b UpSet plot comparing RBPs identified in mESC and different mouse organs by HARD-AP. c Venn diagram comparing RBPs identified in different mouse samples by HARD-AP. d Matrix bubble plot showing enrichments of molecular function GO terms of in RBPs of indicated samples. The GO enrichment analysis used the two-sided Fisher’s exact test with the p-value adjusted using the Bonferroni correction for multiple testing. e Venn diagram comparing the mouse RBPome identified using HARD-AP (mouse RBPs_HARD) and all human RBPome (human RBPs_All) to their indicated orthologs. f Heatmap of the hierarchical clustering analysis using normalized ion intensities from indicated samples. g Top GO terms over-represent in tissue- and cell-enriched RBPs identified by HARD-AP. The GO enrichment analysis used the two-sided Fisher’s exact test with the p -value adjusted using the Bonferroni correction for multiple testing. h Relative levels of indicated proteins in different samples, which are calculated from the ion intensities of three independent biological samples. Data are means ± SD. HARD and EGFP represent proteins isolated by the HARD beads and EGFP beads respectively. i Western blot analysis showing the endogenous protein levels of Bcr, Prkar1a and Mylk3 in different mouse organs and mESC. This experiment was repeated once with similar results. j Western blot analysis for indicated proteins in indicated organ lysates after capture under indicated conditions. This experiment was repeated once with similar results. k Images of Cy5-RNA incubation signal of the human orthologs of Bcr, Prkar1a and Mylk3 on the protein microarray. Source data for ( a – c ) are provided as a Source Data file.

Article Snippet: Anti-DNMT1(Sino Biological, cat#100780-T10, 1:1000), anti-PTBP1(Sino Biological, cat#101043-T46, 1:1000), anti-Histone H3(Sino Biological, cat#100005-MM01, 1:10,000), anti-PSPC1(Proteintech, cat#16714-1-AP, 1:1000), anti-NONO(Proteintech, cat#11058-1-AP, 1:1000), anti-Bcr(Proteintech, cat#22585-1-AP, 1:1000), anti-Prkar1a(Proteintech, cat#20358-1-AP, 1:1000), anti-Mylk3(Proteintech, cat#21527-1-AP, 1:1000), anti-Csrp1(ABclonal, cat#A19842, 1:1000), anti-V5 tag(Sino Biological, cat#100378-T36, 1:2000), anti-Gapdh(Proteintech, cat#60004-1-Ig, 1:10000), anti-β-tubulin (Proteintech, cat#10068-1-AP, 1:10,000), Goat anti-mouse IgG (H + L) HRP (Sino Biological, cat#SSA007, 1:1000), Goat anti-rabbit IgG (H + L) HRP (Sino Biological, cat#SSA004, 1:1000).

Techniques: Two Tailed Test, Isolation, Western Blot, Incubation, Microarray