Journal: Oncotarget

Article Title: Overexpression of C16orf74 is involved in aggressive pancreatic cancers

doi: 10.18632/oncotarget.10912

Figure Lengend Snippet: A. Western blot analysis of the expression levels of C16orf74 in pancreatic cancer cell lines. Control: Flag-tagged C16orf74-overexpressed diluted cell lysate. B. Phosphorylated form (arrow) of endogenous C16orf74 in KLM-1 cells, as examined by Western blot analysis using an anti-C16orf74 polyclonal antibody. The upper band disappeared when the cell lysate was incubated with lambda phosphatase (PPase (+)). C. Phosphorylation at threonine 44 (T44) of C16orf74. Flag-tagged wild type (WT), T41A and T44A mutants of C16orf74 were used to transfect COS-7 cells. The phosphorylated form of wild-type C16orf74 (arrow) was disappeared in the T44A mutant. D. Immunocytochemical analysis in a pancreatic cancer cell line (PK-1) using the anti-C16orf74 antibody, demonstrating the plasma membrane localization of endogenous C16orf74 (Green). DAPI staining is shown in blue.

Article Snippet: In the PPP3CA interaction assay, the KLM-1 cell lysate was incubated with lambda protein phosphatase, immunoprecipitated by mouse monoclonal PPP3CA (sc-17808, Santa Cruz) and immunoblotted with rabbit polyclonal C16orf74, as in the western blot analysis.

Techniques: Western Blot, Expressing, Control, Incubation, Phospho-proteomics, Mutagenesis, Clinical Proteomics, Membrane, Staining

Journal: Oncotarget

Article Title: Overexpression of C16orf74 is involved in aggressive pancreatic cancers

doi: 10.18632/oncotarget.10912

Figure Lengend Snippet: A. In vitro exogenous association of C16orf74 and PPP3CA. The Flag-tagged C16orf74 construct or vector alone was cotransfected with a myc-tagged PPP3CA construct into HEK293 cells. Cell lysates were immunoprecipitated using mouse anti-Flag antibody (left) or anti-myc antibody (right). Immunoblotting of the immunoprecipitates with rabbit anti-Flag or anti-myc antibodies revealed a specific interaction between the phosphorylated form of C16orf74 (arrow) and PPP3CA. B. In vitro endogenous association of C16orf74 and PPP3CA from Capan-1 pancreatic cancer cells, which endogenously express high levels of both C16orf74 and PPP3CA. Capan-1 cell lysates were immunoprecipitated using anti-C16orf74 antibody (left) or anti- PPP3CA antibody (right). Immunoblotting of the immunoprecipitates with anti-C16orf74 antibody or anti-PPP3CA antibodies revealed a specific interaction between C16orf74 and PPP3CA. Endogenous PPP3CA interacted with the phosphorylated form of endogenous C16orf74 (arrow). C. Interactions of wild-type C16orf74 (WT) and mutants of C16orf74 with PPP3CA, as assessed by IP analysis. Expression vectors for myc-His-tagged PPP3CA and Flag-tagged C16orf74 constructs were doubly transfected into HEK293T cells. C16orf74 (anti-Flag) was IP, and the indicated molecules were immunoblotted (IB) in western blot analysis. WT, replacement (T44A; non-phosphorylated form of C16orf74) and deletion mutants (∆PDIIIT; deletion mutant of PPP3CA binding motif) were analyzed. PPP3CA bound to wild-type C16orf74 but not the non-phosphorylated form of C16orf74 or the deletion mutant of the PPP3CA binding motif. D. Subcellular localization of C16orf74 (wild type or ∆PDIIIT) and PPP3CA in mammalian cells. Flag-tagged (green) C16orf74 (wild type or ∆PDIIIT) and myc-tagged (red) PPP3CA constructs were cotransfected into COS-7 cells and subjected to immunocytochemical staining. Flag-C16orf74 (wild type) and myc-PPP3CA colocalized on the under the cytoplasmic membrane of COS-7 cells (yellow), but Flag-C16orf74 (∆PDIIIT) did not colocalize with myc-PPP3CA, which was present diffusely in the cytoplasm. E. Interactions of endogenous C16orf74 with PPP3CA as assessed by IP analysis. The phosphorylated form (arrow) of endogenous C16orf74 in KLM-1 cells, as examined by western blot analysis using an anti-C16orf74 polyclonal antibody. Pre IP (left; non-immunoprecipitated by PPP3CA), the phosphorylated form of C16orf74 (upper band) disappeared when the cell lysate was incubated with lambda phosphatase (PPase (+)). Immunoprecipitation by PPP3CA (right) revealed that the phosphorylated form of C16orf74 (upper band) interacted with PPP3CA, whereas the non- phosphorylated form of C16orf74 did not. F. Invasion activity of wild-type C16orf74 (WT) and the two mutants (T44A: non-phosphorylated form of C16orf74; and ∆PDIIIT, deletion mutant of the PPP3CA binding motif). The WT-C16orf74 expression vector, T44A-C16orf74 expression vector, ∆PDIIIT-C16orf74 expression vector, and Mock vector were each transfected into NIH3T3 cells. The Matrigel invasion assay revealed an enhanced cell number for WT-C16orf74-over-expressing cells (3.4-fold, * P = 0.013) but not so enhanced for ∆PDIIIT-C16orf74-over-expressing cells (1.4-fold, ** P = 0.017) or T44A-C16orf74-over-expressing cells (2.3-fold,*** P = 0.038).

Article Snippet: In the PPP3CA interaction assay, the KLM-1 cell lysate was incubated with lambda protein phosphatase, immunoprecipitated by mouse monoclonal PPP3CA (sc-17808, Santa Cruz) and immunoblotted with rabbit polyclonal C16orf74, as in the western blot analysis.

Techniques: In Vitro, Construct, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Transfection, Mutagenesis, Binding Assay, Staining, Membrane, Incubation, Activity Assay, Invasion Assay

Journal: Cell

Article Title: Structural insights into the process of GPCR-G protein complex formation

doi: 10.1016/j.cell.2019.04.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains E. coli cells BL21(DE3) CWBIO CW0809S E. coli cells TOP10 CWBIO CW0807S E. coli cells Rossetta 2 (DE3) EMD Millipore 70954 E. coli cells DH10Bac Invitrogen 10361012 Chemicals, Peptides, and Recombinant Proteins Benzamidine Sigma Cat#B6506 Leupeptin Sigma Cat#L2884 n-dodecyl-beta-D-maltopyranoside (DDM) Anatrace Cat#D310 Lauryl Maltose Neopentyl Glycol (MNG) Anatrace Cat#NG310 Cholesterol hemisucinate (CHS) Sigma Cat#C6512 ANTI-FLAG M1 Agarose Affinity Gel Sigma-Aldrich Cat#A4596 1-Oleoyl-rac-glycerol (monoolein) Sigma Cat#M7765 Cholesterol Sigma Cat#C8667 POPG Avanti Cat# 840457 FLAG peptide Sigma-Aldrich Cat# F3290 Alprenolol Sigma-Aldrich Cat# A8676 BI-167107 Custom N/A Isoproterenol Tocris Cat# 1747 GDP Sigma Cat# 7127 ESF921 culture medium Expression Systems Cat# 96–001 Monobromobimane Invitrogen Cat# M1378 FBS VWR Cat#97068–085 PNGase F New England Biolabs Cat# P0708 Lambda Protein Phosphatase (Lambda PP) New England Biolabs Cat# P0753 TCEP Sigma-Aldrich Cat# C4706 Antarctic Phosphatase New England Biolabs Cat# M0289 CIP New England Biolabs Cat# M0290 DpnI New England Biolabs Cat# R0176 Dihydroalprenolol hydrochloride, Levo-[ring, propyl- 3 H(N)] PerkinElmer Cat# NET720001MC Polyethylenimine Sigma Cat# 408727 cOmplete™, EDTA-free Protease Inhibitor Cocktail Roche Cat# 30307800 CK28 beads Bertin Corp. Cat# 03961CK28 GF/B unifilters, Whatmann PerkinElmer Cat# 6005177 Deposited Data β2AR-T4L-GsCT-CC structure This paper PDB: 6E67 Gs GDP structure This paper PDB: 6EG8 Experimental Models: Cell Lines Insect cell line Sf9 Expression Systems N/A Insect cell line High Fives (Tni) Expression Systems N/A BHK-21 cell line ATCC Cat# ATCC® CCL-10™ Recombinant DNA pfastbac-wtβ2AR This study N/A pfastbac-mutβ2AR This study N/A pfastbac-Gαs/βγ This study N/A Pfastbac-mutGαs/βγ This study N/A pET28a-ApoA1 Denisov et al., 2004 Addgene Plasmid# 20060 pET28a-Gαs C3S This study N/A pVLdual_Gβγ C68S This study N/A pcDNA3.1(+)-β2AR This study N/A pcDNA3.1(+)-β2AR-cpep This study N/A pcDNA3.1(+)-β2AR-cpep mutants This study N/A Software and Algorithms COOT Emsley et al., 2010 www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot XDS Kabsch, 2010 http://xds.mpimf-heidelberg.mpg.de/ Phaser McCoy et al., 2007 http://www.ccp4.ac.uk Phenix Adams et al., 2010 https://www.phenix-online.org PyMOL Schrodinger LLC https://www.pymol.org/2/ KAMO Yamashita et al., 2018 https://github.com/keitaroyam/yamtbx Prism v.6.0 GraphPad Software, Inc. https://www.graphpad.com Open in a separate window KEY RESOURCES TABLE An active β2AR structure was obtained by fusion with residues 381–394 of Gs (GsCT).

Techniques: Recombinant, Expressing, Protease Inhibitor, Plasmid Preparation, Software

Journal: PLoS Pathogens

Article Title: Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis

doi: 10.1371/journal.ppat.1010057

Figure Lengend Snippet: (A) Illustration of wild-type precore protein (p25-WT) and mutant/HA-tagged p25 as well as its proteolytically processed products p22 and p17 that are expressed by pXF3H-derived plasmids (see for detailed description). Red arrow indicates the mutation of Cp starting codon (AUG) to AUA. The gray box indicates the insertion of HA tag at the immediate upstream of Cp starting codon. (B) HepG2 cells were transfected with pXF3H-p25-WT and derived plasmid expressing p25 with the indicated single amino acid substitution. The cells were harvested at 48 h post transfection. Intracellular p25-derived proteins were detected by Western blot assay with an antibody recognizing the last 14 amino acid residues at the C-terminus of precore protein and Cp (anti-HBc-170A). HBeAg in culture media was detected by CLIA and results (mean ± SD) from three independent experiments were analyzed by two-tailed Student’s t-test (unpaired), ***: P < 0.001. (C) HepG2 cells were transfected with a pXF3H-derived plasmid expressing the indicated p25 proteins or pCMV-HBc expressing Cp. Intracellular p25-derived proteins or Cp were detected by Western blot assay with an anti-HBc-170A as the primary antibody. The lysate from AML12HBVpolY63F cells was analyzed in parallel to serve as the control of hyper- and hypo-phosphorylated Cp. (D) HepG2 cells were transfected with pXF3H-p25HA, harvested at 48 h post transfection and lysed with core DNA lysis buffer. Aliquots of the cell lysate were mock-treated or treated with Lambda phosphatase at 30°C for 40 min. HBV p22 in the reactions were detected by Western blot assay with anti-HBc-170A. ( E ) HepG2 cells were transfected with a plasmid expressing the indicated precore protein and harvested at 48 h post transfection. The cells were lysed by 1× LDS lysis buffer (contained 2.5% B-ME) and the lysates were resolved by electrophoresis in NuPAGE 12% Bis-Tris Protein Gel (upper panel) and 12% phos-tag gel (lower panel). After blotting onto membranes, p22 were detected by anti-HBc-170A as primary antibody. β-actin served as a loading control.

Article Snippet: HepG2 cells transfected with desired plasmids were lysed by core DNA lysis buffer, 40 μl lysed samples were incubated with 1μl Lambda phosphatase (NEB, MA, USA), 5 μl 10 ×NEB buffer 3.1 and 4 μl nuclease-free water for 40 min at 30°C.

Techniques: Mutagenesis, Derivative Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Two Tailed Test, Lysis, Electrophoresis