Journal: Cell Chemical Biology

Article Title: Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction

doi: 10.1016/j.chembiol.2017.06.015

Figure Lengend Snippet: Validation of Flavidin Thermostability and Ligand Binding (A) Flavidin thermostability after heating for 3 min in PBS at the indicated temperature and then analysis of tetramer integrity by SDS-PAGE with Coomassie blue staining. C is a control boiled in SDS before loading. (B) Biotin-4-fluorescein association rates for Flavidin with or without 635P-NHS labeling (mean ± 1 SD, n = 9). (C) Biotin-4-fluorescein dissociation rates for Flavidin with or without 635P-NHS labeling (mean of triplicate ± 1 SD). See also Figure S4 .

Article Snippet: Requests for plasmids for Flavidin (pET21-Flavidin, https://www.addgene.org/89881/ ) and K121R mutant (pET21-Streptavidin-K121R, https://www.addgene.org/89880/ ) may also be made from Addgene.

Techniques: Biomarker Discovery, Ligand Binding Assay, SDS Page, Staining, Control, Labeling

Journal: Cell Chemical Biology

Article Title: Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction

doi: 10.1016/j.chembiol.2017.06.015

Figure Lengend Snippet:

Article Snippet: Requests for plasmids for Flavidin (pET21-Flavidin, https://www.addgene.org/89881/ ) and K121R mutant (pET21-Streptavidin-K121R, https://www.addgene.org/89880/ ) may also be made from Addgene.

Techniques: Virus, Recombinant, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Sequencing, Software

Journal: MethodsX

Article Title: Optimization of production of recombinant gamma-tubulin in bacteria

doi: 10.1016/j.mex.2021.101517

Figure Lengend Snippet: Optimizing the conditions for expressing recombinant TUBG1: (A) a scheme for the optimization of culture conditions for the expression of TUBG1 using ampicillin (Amp) as a selection marker in transformed E . coli. In short, a selected E. coli colony carrying a vector containing the TUBG1 and Amp genes is cultured overnight (ON) in Luria–Bertani liquid supplemented with ampicillin. Then, the bacteria are diluted and cultured until the optical density measured at a wavelength of 600 nm (OD 600 ) reaches 0.6 to 0.8. The expression of recombinant TUBG1 is induced with isopropyl-1-thio-β-D-galactopyranoside (IPTG) during the indicated times and conditions. GST-TUBG1 (B) and TUBG1-His 6 (C) were induced in E. coli DH5α containing the pGEX2T- TUBG1 vector with 0.2 mM IPTG (B) and in E. coli BL21 carrying the pET- TUBG1 -His 6 with 1 mM IPTG (C), respectively. Thereafter, the bacteria were incubated under the following conditions: 1 h at 37 °C (1 h, 37 °C), overnight incubation at room temperature (ON, RT), and 1 h at 37 °C followed by overnight incubation at room temperature (1 h, 37 °C ON, RT). (B and C) The expression of recombinant TUBG1 was analyzed by Western blot (WB) using an anti-TUBG1 antibody. The highest yield of GST-TUBG1 was obtained upon stimulation with 0.2 mM IPTG for 1 h at 37 °C followed by overnight incubation at room temperature (B), whereas the highest yield of TUBG1-His 6 was reached after stimulation with 1.0 mM IPTG for 1 h at 37 °C (C). (B and C) The graphs illustrate densitometric analysis of the TUBG1 content in the Western blots presented (mean ± SD; N = 3, ** P < 0.01, * P < 0.05).

Article Snippet: Purification of GST- and His 6 -tagged human TUBG1 from bacteria lysates Step 1: Optimization of the culture conditions with small-scale expression cultures Materials ▪ pGEX2T- TUBG1 (Addgene Plasmid # 171967) ▪ pET21- TUBG1 (Addgene Plasmid # 101825) ▪ E. coli DH5α (gives high protein yield from pGEX2T- TUBG1 plasmid; Thermo Fisher Scientific, cat. no. 18265017) ▪ E. coli BL21([DE3] optimized for protein expression from the T7 promoter included in a pET21 plasmid; Thermo Fisher Scientific, cat. no. C600003) ▪ Sterile Luria–Bertani liquid (LB) medium (Merck, cat. no. L2542) ▪ Sterile LB agar plates (Sigma-Aldrich, cat. no. L5667) ▪ Ampicillin (Thermo Fisher Scientific, cat. no. 11593027) dissolved in sterile, deionized distilled water ▪ Sterile 14 ml polypropylene round-bottom tubes (Fisher scientific; Falcon, cat. no. 10384641) ▪ Isopropyl-β-D- thiogalactoside (IPTG; Sigma-Aldrich, cat. no I6758) dissolved in sterile, deionized distilled water ▪ Phenylmethylsulphonyl fluoride (PMSF; Sigma-Aldrich, cat. no 10837091001) dissolved according to the manufacturer's instructions ▪ Anti-TUBG (1:1000; Sigma-Aldrich, cat. no T3320) ▪ Sterile phosphate-buffered saline (PBS; Sigma-Aldrich, cat. no. P4417) ▪ 1.5 ml microcentrifuge tubes (Sigma-Aldrich, cat. no. Z336777) ▪ Shaking incubator for culture growth (Merck, cat. no. CLS6791) ▪ Soniprep 150 Plus with exponential probe (240V; MSE, cat. no. MSS150.CX4.5) ▪ 5X loading buffer: 625 mM TRIS pH 6.5 (Sigma-Aldrich, cat. no. 10812846001), 10% (w/v) sodium dodecyl sulfate (SDS; Sigma-Aldrich, cat. no. GE17-1313-01), 25% (v/v) glycerol (Sigma-Aldrich, cat. no. G5516), 0.005% (w/v) bromophenol blue (Sigma-Aldrich, cat. no. GE17-1329-01), 250 mM dithiothreitol (Sigma-Aldrich, cat. no. D0632), and deionized distilled water Note that the preceding list includes only the necessary bacteria strains and non-standard laboratory equipment.

Techniques: Expressing, Recombinant, Selection, Marker, Transformation Assay, Plasmid Preparation, Cell Culture, Bacteria, Incubation, Western Blot

Journal: MethodsX

Article Title: Optimization of production of recombinant gamma-tubulin in bacteria

doi: 10.1016/j.mex.2021.101517

Figure Lengend Snippet: Purification of GST-TUBG1 and removal of the GST tag: a scheme of the production and affinity purification of GST-TUBG from E. coli DH5α carrying the pGEX2T- TUBG1 vector and using ampicillin (Amp) as a selection marker. In short, the E. coli colony from a pre-selected colony is cultured overnight (ON) in Luria–Bertani liquid supplemented with ampicillin. Then, the bacteria are diluted and cultured until the optical density measured at a wavelength of 600 nm (OD 600 ) reaches 0.6 to 0.8. The expression of recombinant TUBG1 is induced with 0.2 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 1 h at 37 °C followed by overnight incubation at room temperature. The bacteria are harvested and lysed by sonication. After preclearing the lysates by centrifugation (centrif.), the recombinant GST-TUBG1 in the resulting bacterial lysates (lysates) is affinity purified with Glutathione Sepharose 4B beads. The GST tag is thereafter cleaved with thrombin after removal of PMSF (Dirty beads) or after extensive washing (Washed beads). The expression of recombinant TUBG1 and the efficient removal of GST were analyzed by SDS-page stained with Gelcode Blue and by Western blotting (WB) using an anti-TUBG1 antibody and anti-GST antibodies. The color of the arrows shows the sequence of events in the scheme (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Article Snippet: Purification of GST- and His 6 -tagged human TUBG1 from bacteria lysates Step 1: Optimization of the culture conditions with small-scale expression cultures Materials ▪ pGEX2T- TUBG1 (Addgene Plasmid # 171967) ▪ pET21- TUBG1 (Addgene Plasmid # 101825) ▪ E. coli DH5α (gives high protein yield from pGEX2T- TUBG1 plasmid; Thermo Fisher Scientific, cat. no. 18265017) ▪ E. coli BL21([DE3] optimized for protein expression from the T7 promoter included in a pET21 plasmid; Thermo Fisher Scientific, cat. no. C600003) ▪ Sterile Luria–Bertani liquid (LB) medium (Merck, cat. no. L2542) ▪ Sterile LB agar plates (Sigma-Aldrich, cat. no. L5667) ▪ Ampicillin (Thermo Fisher Scientific, cat. no. 11593027) dissolved in sterile, deionized distilled water ▪ Sterile 14 ml polypropylene round-bottom tubes (Fisher scientific; Falcon, cat. no. 10384641) ▪ Isopropyl-β-D- thiogalactoside (IPTG; Sigma-Aldrich, cat. no I6758) dissolved in sterile, deionized distilled water ▪ Phenylmethylsulphonyl fluoride (PMSF; Sigma-Aldrich, cat. no 10837091001) dissolved according to the manufacturer's instructions ▪ Anti-TUBG (1:1000; Sigma-Aldrich, cat. no T3320) ▪ Sterile phosphate-buffered saline (PBS; Sigma-Aldrich, cat. no. P4417) ▪ 1.5 ml microcentrifuge tubes (Sigma-Aldrich, cat. no. Z336777) ▪ Shaking incubator for culture growth (Merck, cat. no. CLS6791) ▪ Soniprep 150 Plus with exponential probe (240V; MSE, cat. no. MSS150.CX4.5) ▪ 5X loading buffer: 625 mM TRIS pH 6.5 (Sigma-Aldrich, cat. no. 10812846001), 10% (w/v) sodium dodecyl sulfate (SDS; Sigma-Aldrich, cat. no. GE17-1313-01), 25% (v/v) glycerol (Sigma-Aldrich, cat. no. G5516), 0.005% (w/v) bromophenol blue (Sigma-Aldrich, cat. no. GE17-1329-01), 250 mM dithiothreitol (Sigma-Aldrich, cat. no. D0632), and deionized distilled water Note that the preceding list includes only the necessary bacteria strains and non-standard laboratory equipment.

Techniques: Purification, Affinity Purification, Plasmid Preparation, Selection, Marker, Cell Culture, Bacteria, Expressing, Recombinant, Incubation, Sonication, Centrifugation, SDS Page, Staining, Western Blot, Sequencing

Journal: Hepatology Communications

Article Title: Prohibitin 1 Acts As a Negative Regulator of Wingless/Integrated‐Beta‐Catenin Signaling in Murine Liver and Human Liver Cancer Cells

doi: 10.1002/hep4.1257

Figure Lengend Snippet: Increased binding of E2F1 to the Wnt10a promoter in Phb1 KO livers. (A,B) Phb1 silencing in mouse primary hepatocytes induced Wnt7a and Wnt10a. Total RNA and protein were prepared and subjected to qPCR and western blot, respectively. Relative expression was represented as fold induction compared to NC; n = 3; * P < 0.05 versus NC. (C) Predicted E2F1 binding sites in the mouse Wnt10a (Accession ID, U61969.1) sequence as determined by ALGGEN PROMO Transcription Factor bindings prediction software. Transcription start site is shown as bold with GGC underlined. Underlined sequences represent primer sequences used for ChIP PCR assay. (D,E) E2F1 binding to the promoter was determined by ChIP assay of chromatin prepared from 3‐week‐old Phb1 KO and WT mice livers. ChIP assay was performed as described in the Materials and Methods. Relative target site occupancy is represented as fold over WT control. Results represent mean ± SEM from six WT and KO livers in duplicate experiments; * P < 0.05 versus WT.

Article Snippet: E2F1 plasmid was purchased from Addgene (Cambridge, MA).

Techniques: Binding Assay, Western Blot, Expressing, Sequencing, Software, Control

Journal: Hepatology Communications

Article Title: Prohibitin 1 Acts As a Negative Regulator of Wingless/Integrated‐Beta‐Catenin Signaling in Murine Liver and Human Liver Cancer Cells

doi: 10.1002/hep4.1257

Figure Lengend Snippet: WNT9A induction by PHB1 silencing is E2F1 dependent. (A) Effect of PHB 1 silencing on WNT ligand induction in HepG2 cells. HepG2 cells were forward transfected with NC or PHB1 siRNA for 24 hours and 48 hours. Relative expression of WNT9A, WNT10A, WNT16, and PHB1 was compared to the NC. Results represent mean ± SEM from three independent experiments; # P < 0.001, * P < 0.05 versus NC. (B ) Effect of PHB1 and E2F1 cosilencing for 48 hours on WNT9A expression in HepG2 cells. Results represent mean ± SEM from three independent experiments performed in duplicates; # P < 0.001 for si PHB1 versus NC, si PHB1 versu s si PHB1 +si E2F1 . (C ) Effect of PHB1 cosilencing with E2F1 overexpression on WNT9A levels in HepG2 cells. Results represent mean ± SEM from three independent experiments performed in duplicates. # P < 0.001 for si PHB1 versus NC, EV versus E2F1 OE, si PHB1 versus si PHB1 + E2F1 OE, E2F1 OE versus si PHB1 + E2F1 OE. (D) Effect of E2F1 OE for 48 hours in conjunction with 72 hours of PHB1 silencing on phosphorylation of GSK3beta Ser9 . Data represent two independent experiments. (E) Effect of E2F1 OE for 48 hours in conjunction with 72 hours of PHB1 silencing on CCND1 mRNA levels. Data represent three independent experiments; * P < 0.05 versus NC/EV, # P < 0.05 versus si PHB1 and E2F1 OE. (F) Effect of PHB1 silencing and E2F1 overexpression as in (E) on the expression of anti‐apoptotic protein SURVIVIN in HepG2 cells. Data represent two independent experiments.

Article Snippet: E2F1 plasmid was purchased from Addgene (Cambridge, MA).

Techniques: Transfection, Expressing, Over Expression, Phospho-proteomics

Journal: Hepatology Communications

Article Title: Prohibitin 1 Acts As a Negative Regulator of Wingless/Integrated‐Beta‐Catenin Signaling in Murine Liver and Human Liver Cancer Cells

doi: 10.1002/hep4.1257

Figure Lengend Snippet: PHB1 silencing leads to increased E2F1 binding to the WNT9A promoter in HepG2 cells. (A) Predicted E2F1 binding sites (in bold) in the human WNT9A (Accession ID, HM015601.1) sequence was determined by ALGGEN PROMO transcription factor bindings prediction software. Transcription start site is gcg underlined in bold. Sequences that are underlined are primer sequences used for the ChIP assay. (B) E2F1 binding to the WNT9A promoter was determined by ChIP assay. Chromatin was prepared from NC and PHB1 ‐silenced HepG2 cells, and ChIP assay was performed as described in Materials and Methods. Relative target site occupancy is represented as fold over NC. Results represent mean ± SEM from three independent experiments in duplicates; * P < 0.05 versus NC. (C) Effect of E2F1 OE for 48 hours in conjunction with 72 hours of PHB1 silencing on E2F1 binding to the WNT9A promoter in HepG2 cells. Results represent mean ± SEM from three to six independent experiments in duplicates; * P < 0.05 versus NC/EV, # P < 0.05 E2F1 OE versus E2F1 OE+si PHB 1. (D) Summary demonstrating the potential role of PHB1 in modulating WNT signaling in Phb1 KO livers and HepG2 cells. PHB1 deletion leads to induction of WNT ligands in an E2F1‐dependent manner. WNT ligands induce downstream activation of WNT‐beta‐catenin signaling through the AKT‐GSK3beta signaling pathway. This results in increased TCF transcriptional activity and cell proliferation. Abbreviations: APC, adenomatous polyposis coli; c‐MYC, Myc Proto‐Oncogene; FzD, Frizzled.

Article Snippet: E2F1 plasmid was purchased from Addgene (Cambridge, MA).

Techniques: Binding Assay, Sequencing, Software, Activation Assay, Activity Assay