Journal: Cell Death & Disease

Article Title: Aldolase B-driven lactagenesis and CEACAM6 activation promote cell renewal and chemoresistance in colorectal cancer through the Warburg effect

doi: 10.1038/s41419-023-06187-z

Figure Lengend Snippet: A Metabolomic analysis based on nuclear magnetic resonance (NMR) showing metabolite levels in CRC cells with or without ALDOB overexpression. Black dashed lines indicate relative metabolite levels in control-treated cells. B Lactate levels in medium from cells with control or ALDOB overexpression. C Transwell-based co-culture system for assessment of cell growth (left panel) and chemoresistance (right panels). D Supplementation of medium from cells with or without ALDOB overexpression for assessment of cell proliferation (left panel) and chemoresistance (right panels). E Western blots demonstrating levels of the indicated proteins in cells with or without ALDOB overexpression. The statistical analysis is displayed in the right panel. F Immunofluorescence assay showing the expression of exogenous ALDOB (ALDOB-MYC) and LDHB in CRC cells transfected with ALDOB expression plasmid. The scale bar represents 20 μm. G Western blots showing the levels of the indicated proteins in cells treated with different concentrations of lactate. H Immunofluorescence assay showing LDHB expression in CRC cells treated with indicated concentrations of lactate. The scale bar represents 20 μm. All P values were obtained using the paired two-tailed Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: A mouse monoclonal antibody targeting the MYC tag (Proteintech, Cat. 60003-2-Ig), and a rabbit monoclonal antibody to LDHB (Proteintech, Cat. 14824-1-AP) were used at a 1:200 dilution.

Techniques: Nuclear Magnetic Resonance, Over Expression, Control, Co-Culture Assay, Western Blot, Immunofluorescence, Expressing, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Plant Biotechnology Journal

Article Title: AaSIZ1 ‐Mediated SUMOylation of AaMYB31 Positively Regulates Freezing Tolerance in Actinidia arguta

doi: 10.1111/pbi.70620

Figure Lengend Snippet: AaSIZ1‐mediated SUMOylation of AaMYB31s. (A) In vitro SUMOylation of AaMYB31s. SUMOylated GST‐AaMYB31.1 and GST‐AaMYB31.2 were detected with anti‐GST and anti‐SUMO1 antibodies. (B) In vivo SUMOylation of AaMYB31s in Actinidia arguta protoplasts. AaMYB31.1‐Myc or AaMYB31.2‐Myc was co‐expressed with FLAG‐SUMO1 GG or FLAG‐SUMO1 AA in Actinidia arguta protoplasts. Myc‐AaMYB31.1 and Myc‐AaMYB31.2 were immunoprecipitated with an anti‐Myc antibody, and the immunoprecipitated proteins were detected with anti‐Myc and anti‐FLAG antibodies. (C) In vivo SUMOylation of AaMYB31s in N. benthamiana . AaMYB31.1‐GFP or AaMYB31.2‐GFP was co‐expressed with FLAG‐SUMO1 GG or FLAG‐SUMO1 AA in N. benthamiana leaves. AaMYB31.1‐GFP and AaMYB31.2‐GFP were immunoprecipitated with an anti‐GFP antibody, and the immunoprecipitated proteins were detected with anti‐GFP and anti‐FLAG antibodies. (D) In planta SUMOylation of AaMYB31s. Total proteins were extracted from freshly harvested leaves of WT Actinidia chinensis , 35S::AaMYB31.1‐GFP , and 35S::AaMYB31.2‐GFP transgenic plants, and the AaMYB31.1‐GFP and AaMYB31.2‐GFP were immunoprecipitated with an anti‐GFP antibody. The immunoprecipitated proteins were detected with anti‐GFP and anti‐SUMO1 antibodies. (E) In vivo SUMOylation of AaMYB31.1‐GFP, AaMYB31.2‐GFP, AaMYB31.1 K76R ‐GFP, and AaMYB31.1 K76R ‐GFP in N. benthamiana leaves. AaMYB31.1‐GFP, AaMYB31.1 K76R ‐GFP, AaMYB31.2‐GFP, or AaMYB31.2 K76R ‐GFP was co‐expressed with FLAG‐SUMO1 GG or FLAG‐SUMO1 AA in N. benthamiana leaves. AaMYB31.1‐GFP and AaMYB31.2‐GFP were co‐expressed with FLAG‐SUMO1 AA as a negative control. AaMYB31.1‐GFP or AaMYB31.2‐GFP was immunoprecipitated with an anti‐GFP antibody, and the immunoprecipitated proteins were detected with anti‐GFP and anti‐FLAG antibodies. (F) In planta SUMOylation of AaMYB31.1‐GFP, AaMYB31.2‐GFP, AaMYB31.1 K76R ‐GFP, and AaMYB31.1 K76R ‐GFP. Total proteins were extracted from freshly harvested leaves of 35S::AaMYB31.1‐GFP , 35S::AaMYB31.2‐GFP , 35S::AaMYB31.1 K76R ‐GFP , and 35S::AaMYB31.1 K76R ‐GFP transgenic plants, and the GFP‐fusion proteins were immunoprecipitated with an anti‐GFP antibody. The immunoprecipitated proteins were detected with anti‐GFP and anti‐SUMO1 antibodies. (G) AaSIZ1 SUMOylated AaMYB31s protein in vitro. SUMOylated GST‐AaMYB31.1 and GST‐AaMYB31.2 were detected with anti‐GST and anti‐SUMO1 antibodies. (H) In vivo AaSIZ1 SUMOylation of AaMYB31s in N. benthamiana . AaMYB31.1‐GFP or AaMYB31.2‐GFP and AaSIZ1 were co‐expressed with or without FLAG‐SUMO1 in N. benthamiana leaves. AaMYB31.1‐GFP and AaMYB31.2‐GFP were immunoprecipitated with an anti‐GFP antibody, and the immunoprecipitated proteins were detected with anti‐GFP and anti‐FLAG antibodies. (I) The SUMOylated of AaMYB31s in the 35S::AaSIZ1‐GFP transgenic plants. Total proteins were extracted from freshly harvested leaves of WT Actinidia chinensis and 3 5S::AaSIZ1‐GFP transgenic plants and incubated with GST‐AaMYB31.1 or GST‐AaMYB31.2. SUMOylated GST‐AaMYB31.1 and GST‐AaMYB31.2 were detected with anti‐GST antibody. (J) AaSIZ1 mediates the SUMOylation of AaMYB31s in Actinidia arguta protoplasts. Myc‐AaMYB31.1 or Myc‐AaMYB31.2 was co‐expressed with FLAG‐SUMO1 in Actinidia arguta protoplasts. Myc‐AaMYB31.1 and Myc‐AaMYB31.2 were immunoprecipitated with an anti‐Myc antibody, and the immunoprecipitated proteins were detected with anti‐Myc and anti‐FLAG antibodies.

Article Snippet: Then the protein was immunoprecipitated with anti‐GFP (AE012, ABclonal; dilution 1:2000) and anti‐Myc (AE010, ABclonal; dilution 1:2000) antibodies, and the immunoprecipitated proteins were detected with anti‐Myc, anti‐FLAG and anti‐GFP antibodies.

Techniques: In Vitro, In Vivo, Immunoprecipitation, Transgenic Assay, Negative Control, Incubation

Journal: Plant Biotechnology Journal

Article Title: AaSIZ1 ‐Mediated SUMOylation of AaMYB31 Positively Regulates Freezing Tolerance in Actinidia arguta

doi: 10.1111/pbi.70620

Figure Lengend Snippet: AaSIZ1‐mediated SUMOylation of AaMYB31s enhances their protein stability. (A, B) The protein degradation of AaMYB31s in the AaMYB31.1‐GFP and AaMYB31.1 K76R ‐GFP , or AaMYB31.2‐GFP and AaMYB31.1 K76R ‐GFP transgenic plants. Total proteins were extracted from freshly harvested leaves of AaMYB31.1‐GFP and AaMYB31.1 K76R ‐GFP , or AaMYB31.2‐GFP and AaMYB31.1 K76R ‐GFP transgenic plants. AaMYB31.1‐GFP, AaMYB31.1 K76R ‐GFP, AaMYB31.2‐GFP, and AaMYB31.2 K76R ‐GFP were detected with anti‐GFP antibody. ACTIN was used as an internal control. (C, D) In vivo protein degradation of AaMYB31s in N. benthamiana . 35S::GFP or 35S::AaSIZ1‐GFP was co‐expressed with Myc‐AaMYB31s and HA‐RFP in N. benthamiana leaves, and the total protein was extracted. These proteins were treated with DMSO (−MG132) or 50 μM MG132 for the indicated periods of time and sampled simultaneously to detect. Myc‐AaMYB31.1 and Myc‐AaMYB31.2 were detected with anti‐Myc antibody. HA‐RFP was used as an internal control. (E, F) The protein degradation of AaMYB31s in the 35S::AaSIZ1‐GFP transgenic plants. Total proteins were extracted from freshly harvested leaves of WT Actinidia chinensis and 3 5S::AaSIZ1‐GFP transgenic plants and incubated with GST‐AaMYB31.1 or GST‐AaMYB31.2. These proteins were treated with DMSO (−MG132) or 50 μM MG132 for the indicated periods of time and sampled simultaneously to detect. GST‐AaMYB31.1 and GST‐AaMYB31.2 were detected with anti‐GST antibody. ACTIN was used as an internal control.

Article Snippet: Then the protein was immunoprecipitated with anti‐GFP (AE012, ABclonal; dilution 1:2000) and anti‐Myc (AE010, ABclonal; dilution 1:2000) antibodies, and the immunoprecipitated proteins were detected with anti‐Myc, anti‐FLAG and anti‐GFP antibodies.

Techniques: Transgenic Assay, Control, In Vivo, Incubation

Journal: Journal of Biological Chemistry

Article Title: Four-and-a-Half LIM Domain Proteins Inhibit Transactivation by Hypoxia-inducible Factor 1

doi: 10.1074/jbc.m111.278630

Figure Lengend Snippet: FIGURE 1. FHL2 interacts with HIF-1. A, FLAG-HIF-1 co-immunoprecipitates with FHL2. 293T cells were co-transfected with expression vectors encoding FLAG-HIF-1 and either EV or vector encoding Myc epitope-tagged FHL2. At 24 h post-transfection, the cells were lysed and the lysates were immunoprecipi- tated with anti-Myc antibody. IP products and cell lysates were subjected to WB with anti-Myc or anti-FLAG antibody. B, endogenous FHL2 co-immunopre- cipitates with endogenous HIF-1. Hep3B cells were exposed to 1% O2 for 6 h, lysed, and subjected to immunoprecipitation with anti-IgG or anti-FHL2 antibody. IP products were subjected to WB with anti-FHL2 or anti-HIF-1 antibody. C, the location of the basic helix-loop-helix domain (bHLH), Per-Arnt-Sim homology domain (PAS), O2-dependent degradation domain (ODDD), inhibitory domain (ID), and C-TAD of HIF-1 are shown. Sites of prolyl (P) and asparaginyl (N) hydroxylation are indicated. D and E, FHL2 interacts specifically and directly with HIF-1(429–608). Purified GST and GST fusion proteins containing the indicated amino acid residues of HIF-1 were incubated with in vitro transcribed, in vitro translated, and 35S-labeled FHL2, captured with glutathione- Sepharose beads and analyzed by SDS-PAGE and autoradiography (top panels) or by WB with GST antibody (bottom panel).

Article Snippet: Antibodies used in WB assays were: GST (GE Healthcare); V5 (Invitrogen); FLAG (Sigma); -actin (Santa Cruz); Myc epitope, CBP, FHL1, FHL2, and HIF-2 (Novus Biologicals); and HIF-1 and p300 (BD Biosciences).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Purification, Incubation, In Vitro, Labeling, SDS Page, Autoradiography