pkl  (Abcam)

Journal: PLoS ONE

Article Title: Switching of Pyruvate Kinase Isoform L to M2 Promotes Metabolic Reprogramming in Hepatocarcinogenesis

doi: 10.1371/journal.pone.0115036

Figure Lengend Snippet: MiR-122 targeted PKM2 and suppressed PKM2 expression. (A) Seed sequence of miR-122 in the 3UTR of PKM2 was underlined. WT and mutated sequences of PKM2 were inserted into pmiRGLO luciferase vector. (B) Re-expression of miR-122 suppressed luciferase activity of the WT but not the Mut 3UTR of PKM2. EV and sensor sequences served as negative and positive controls, respectively. (C) Re-expression of miR-122 in SMMC-7221 and MHCC-97L cells suppressed PKM2 but not PKL/R expression. (D) Linear regression model demonstrated that PKM2 mRNA expression was inversely correlated with miR-122 expression in HCC (pink) and NT liver tissues (blue). (E) Expression levels of 667 miRNA species in HCC and NT were plotted. Each dot represents one individual miRNA. MiR-122 is the most abundant miRNA in NT liver. (F) MiR-122 expression in NT, HCC, and venous metastases (VM). Data were retrieved from low density microarray in which expressions of 667 miRNA species were compared between NT, HCC, and VM. B , * P

Article Snippet: Antibodies, HCC tissue microarray sections, immunohistochemistry and histology PKM1 (Sigma), phospho-PKM2 (Tyr105) (Cell Signaling Technology, Danvers, MA), PKM2 (Cell Signaling Technology), PKL (Abcam), β actin (Sigma) were used for Western Blotting.

Techniques: Expressing, Sequencing, Luciferase, Plasmid Preparation, Activity Assay, Microarray

Journal: PLoS ONE

Article Title: Switching of Pyruvate Kinase Isoform L to M2 Promotes Metabolic Reprogramming in Hepatocarcinogenesis

doi: 10.1371/journal.pone.0115036

Figure Lengend Snippet: PKM2 expression in human HCC. (A) mRNA expression of PKL, PKM1, and PKM2 in HCC and NT tissues. Values = 2 ΔΔCT , ΔΔCT = (CT PK – CT HPRT ) of HCC - (CT PK – CT HPRT ) of NT. P values, Wilcoxin signed rank test (B) Waterfall plot shows that, at the mRNA level, PKM2 was up-regulated (HCC/NT2 folds) in 29/60 (48.33%) human HCC samples. (C) Representative pictures of IHC staining with antibody against PKM2 in HCC tissue microarray. PKM2 protein was drastically up-regulated in human HCCs as compared to the paired NT tissues. (D) Mann Whitney test showed that PKM2 over-expression was associated with multiple aggressive clinicopathological features in HCC including the presence of tumor microsatellites, presence of venous invasion, and absence of tumor encapsulation. (E) Over-expression of PKM2 in human HCC was associated with poor prognosis. HCC patients were categorized into two groups: PKM2 over-expression and PKM2 normal/under-expression. PKM2 was considered to be over-expressed when HCC/NT2 folds and was considered to be normal/under-expressed otherwise. HCC patients with PKM2 over-expression had a higher 1-year tumor recurrence rate after surgical resection than HCC patients without PKM2 over-expression, 46.667% Vs 25%. (F) Patients with PKM2 over-expression had lower 5-year overall survival rates after surgical resection. P values were calculated by Kaplan-Meir log rank test.

Article Snippet: Antibodies, HCC tissue microarray sections, immunohistochemistry and histology PKM1 (Sigma), phospho-PKM2 (Tyr105) (Cell Signaling Technology, Danvers, MA), PKM2 (Cell Signaling Technology), PKL (Abcam), β actin (Sigma) were used for Western Blotting.

Techniques: Expressing, Immunohistochemistry, Staining, Microarray, MANN-WHITNEY, Over Expression

Journal: Plant Communications

Article Title: The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

doi: 10.1016/j.xplc.2019.100011

Figure Lengend Snippet: LUX and PKL Regulate H3K27me3 Levels at the DOG1 Locus. (A) ChIP assay. PKL antibody was used to pull down different fragments of DOG1 (shown in Figure 2 C) and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. (B) ChIP assay. H3K27me3 antibody was used to pull down different fragments of DOG1 and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. Seedlings were grown under LD conditions for 5 d and samples were harvested at ZT4. Relative enrichment using the H3K27me3 antibody was normalized to that using the H3 antibody. In all experiments, values denote average ± SD of three biological replicates.

Article Snippet: The chromatin samples were immunoprecipitated with anti-PKL , anti-GFP (Abcam, ab1218), or anti-H3K27me3 (Millipore, 07-449) antibodies.

Techniques: Chromatin Immunoprecipitation

Journal: Plant Communications

Article Title: The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

doi: 10.1016/j.xplc.2019.100011

Figure Lengend Snippet: PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.

Article Snippet: The chromatin samples were immunoprecipitated with anti-PKL , anti-GFP (Abcam, ab1218), or anti-H3K27me3 (Millipore, 07-449) antibodies.

Techniques: Y2H Assay, Binding Assay, Activation Assay, Pull Down Assay, Recombinant, Incubation, Immunoprecipitation, Plasmid Preparation

Journal: Plant Communications

Article Title: The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

doi: 10.1016/j.xplc.2019.100011

Figure Lengend Snippet: LUX and ELF3 Affect Circadian Output to Seeds. (A) Relative DOG1 expression in seedlings under free-running conditions. Seedlings were grown under 12 h light/12 h dark for 6 d followed by CL illumination for 24 h. Samples were harvested every 4 h from ZT24. (B) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (6 d after pollination) were harvested every 4 h started from ZT0. (C) Seed germination rate. Col-0, lux , and elf3 plants were grown under LD conditions for 3 weeks and transferred to CL or kept at LD until seed maturation. Germination of freshly harvested seeds in the light was analyzed. (D) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (8 d after pollination) were harvested at ZT8. For (A) , (B) , and (D) , data are the average ± SD of three biological replicates. (E) A working model illustrating the roles of PKL and EC in controlling seed dormancy. LUX binds directly to a specific DNA sequence of DOG1 and recruits PKL to the DOG1 locus through their physical interaction. This interaction increases H3K27me3 levels on DOG1 chromatin, thereby repressing its transcription and leading to reduced seed dormancy. Arrow indicates positive regulation and bar denotes negative regulation.

Article Snippet: The chromatin samples were immunoprecipitated with anti-PKL , anti-GFP (Abcam, ab1218), or anti-H3K27me3 (Millipore, 07-449) antibodies.

Techniques: Expressing, Sequencing