Journal: The Journal of Biological Chemistry

Article Title: A Sensitive Gel-based Method Combining Distinct Cyclophellitol-based Probes for the Identification of Acid/Base Residues in Human Retaining β-Glucosidases *

doi: 10.1074/jbc.M114.593376

Figure Lengend Snippet: Proof-of-principle; ABP labeling of GBA. a, overexpressed myc/His-tagged GBA variants in total COS-7 cells with excess ABP 1 or ABP 2 and subsequently purified via His-tag pulldown. Labeling with ABP 1 in the absence/presence of sodium azide (first, second gel), idem with ABP 2 (third and fourth gel), and GBA detected with α-myc (fifth gel). b, β-epoxide 1 and β-aziridine 2 labeling at different sodium azide concentrations. c, rescue of labeling with small acids. Labeling of wild-type GBA and acid/base mutants E235G and E235Q at pH 5.2 with β-epoxide 1 in the absence or presence of 1 m sodium azide (forms hydrazoic acid (HN3)), 10 m acetic acid (HAc), or 15 m formic acid (HFo), and subsequently purification via His-tag pulldown is shown. Inter-gel comparisons facilitated by excess imiglucerase labeled with 50 fm ABP 1 (asterisk) as positive control.

Article Snippet: Next, the blots were incubated overnight with 1:2,000 diluted primary mouse α-myc mAb (Cell Signaling, b118, 2% (w/v) BSA in TBST) at 4 °C, washing with TBST for 20 min (repeated 6 times), subsequently incubated with 1:10,000 diluted secondary rabbit α-mouse IRD680 (Cell Signaling, 2% (w/v) BSA in TBST) at room temperature, washed with TBST for 20 min (repeated 6 times), and finally, read-out occurred on an Odyssey Infrared Scanner (Li-Cor).

Techniques: Labeling, Purification, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: A Sensitive Gel-based Method Combining Distinct Cyclophellitol-based Probes for the Identification of Acid/Base Residues in Human Retaining β-Glucosidases *

doi: 10.1074/jbc.M114.593376

Figure Lengend Snippet: Cytosolic β-glucosidase GBA3. a, schematic depicting GBA3 with predicted acid/base (left, gray) and nucleophile (right, black). b, catalytic activity of GBA3 variants toward 4MU-, 4NP-, and 2,4DNP-β-d-Glc relative to mock (black, hatched, white columns, respectively). Sodium azide-mediated rescue of 4NP (c)- and 2,4DNP-β-d-Glc (d) hydrolysis by mock (empty plasmid, open circles), wild-type GBA3 (filled circles), E165G (gray triangles), E373Q (gray circles), and E165G/E373G (black squares). e, overexpressed myc/His-tagged GBA3 variants in total COS-7 cells with excess ABP 1 or ABP 2 and subsequently purified via His-tag pulldown. Labeling with ABP 1 in the absence/presence of sodium azide (first and second gels), idem with ABP 2 (third and fourth gel), and GBA3 detected with α-myc (fifth gel). Data are the average of triplicates ± S.D., with one-way analysis of variance significance; ***, p < 0.001.

Article Snippet: Next, the blots were incubated overnight with 1:2,000 diluted primary mouse α-myc mAb (Cell Signaling, b118, 2% (w/v) BSA in TBST) at 4 °C, washing with TBST for 20 min (repeated 6 times), subsequently incubated with 1:10,000 diluted secondary rabbit α-mouse IRD680 (Cell Signaling, 2% (w/v) BSA in TBST) at room temperature, washed with TBST for 20 min (repeated 6 times), and finally, read-out occurred on an Odyssey Infrared Scanner (Li-Cor).

Techniques: Activity Assay, Plasmid Preparation, Purification, Labeling

Journal: The Journal of Biological Chemistry

Article Title: A Sensitive Gel-based Method Combining Distinct Cyclophellitol-based Probes for the Identification of Acid/Base Residues in Human Retaining β-Glucosidases *

doi: 10.1074/jbc.M114.593376

Figure Lengend Snippet: Lactase/phlorizin hydrolase LPH. a, schematic depicting LPH with predicted acid/base (Glu-1065, Glu-1538) and nucleophile (Glu-1273, Glu-1749) for both pockets III and IV. b, catalytic activity of LPH variants toward 4MU-, 4NP-, and 2,4DNP-β-d-Glc relative to mock (black, hatched, white columns, respectively). c, sodium azide-mediated rescue of 2,4DNP-β-d-Glc hydrolysis by mock (empty plasmid, open circles), wild-type LPH (closed circles), E1065G (gray triangles), E1273G (gray circles), E1538G (gray squares), E1749G (gray diamonds), and E1273G/E1749G (black squares). d, glucose liberation from lactose by LPH variants, relative to mock. e, overexpressed myc/His-tagged LPH variants in total COS-7 cells with excess ABP 1 or ABP 2 and subsequently purified via His-tag pulldown. Labeling with ABP 1 in the absence/presence of sodium azide (first and second gel), idem with ABP 2 (third and fourth gel), and LPH detected with α-myc (fifth gel). Data are the average of triplicates ± S.D., with one-way analysis of variance significance; *, p < 0.05; ***, p < 0.001.

Article Snippet: Next, the blots were incubated overnight with 1:2,000 diluted primary mouse α-myc mAb (Cell Signaling, b118, 2% (w/v) BSA in TBST) at 4 °C, washing with TBST for 20 min (repeated 6 times), subsequently incubated with 1:10,000 diluted secondary rabbit α-mouse IRD680 (Cell Signaling, 2% (w/v) BSA in TBST) at room temperature, washed with TBST for 20 min (repeated 6 times), and finally, read-out occurred on an Odyssey Infrared Scanner (Li-Cor).

Techniques: Activity Assay, Plasmid Preparation, Purification, Labeling

Journal: The Journal of Biological Chemistry

Article Title: A Sensitive Gel-based Method Combining Distinct Cyclophellitol-based Probes for the Identification of Acid/Base Residues in Human Retaining β-Glucosidases *

doi: 10.1074/jbc.M114.593376

Figure Lengend Snippet: Klotho, βklotho, and KLPH. a, schematic depicting klotho, βklotho and KLPH with putative acid/bases (gray) and nucleophiles (black) in predicted pockets. b, overexpressed myc/His-tagged klotho, βklotho, KLPH, GBA wild-type, and acid/base-lacking E235G GBA in total COS-7 cells with excess ABP 1 or ABP 2 and purified via His-tag pulldown. Labeling with ABP 1 in the absence/presence of sodium azide (first and second gel), idem with ABP 2 (third and fourth gel), and myc-tagged proteins detected with α-myc (fifth gel).

Article Snippet: Next, the blots were incubated overnight with 1:2,000 diluted primary mouse α-myc mAb (Cell Signaling, b118, 2% (w/v) BSA in TBST) at 4 °C, washing with TBST for 20 min (repeated 6 times), subsequently incubated with 1:10,000 diluted secondary rabbit α-mouse IRD680 (Cell Signaling, 2% (w/v) BSA in TBST) at room temperature, washed with TBST for 20 min (repeated 6 times), and finally, read-out occurred on an Odyssey Infrared Scanner (Li-Cor).

Techniques: Purification, Labeling

Journal: The Journal of Biological Chemistry

Article Title: A Sensitive Gel-based Method Combining Distinct Cyclophellitol-based Probes for the Identification of Acid/Base Residues in Human Retaining β-Glucosidases *

doi: 10.1074/jbc.M114.593376

Figure Lengend Snippet: Non-lysosomal β-glucosidase GBA2. a, catalytic activity of LPH variants toward 4MU-, 4NP-, and 2,4DNP-β-d-Glc relative to mock (black, hatched, white columns, respectively). b, sodium azide-mediated rescue of 2,4DNP-β-d-Glc hydrolysis by mock (empty plasmid, open circles), wild-type GBA2 (filled circles), E527G (filled triangles), D677G (dark gray circles), D659G (dark gray squares), D663G (dark gray diamonds), E667G (light gray triangles), D673G (light gray circles), and E527G/D677G (filled squares). c, overexpressed myc/His-tagged GBA2 variants in total COS-7 cells with excess ABP 1 or ABP 2 and subsequently purified via His-tag pulldown. Labeling with ABP 1 in the absence/presence of sodium azide (first, second gel), idem with ABP 2 (third, fourth gel), and GBA2 detected with α-myc (fifth gel). Data are the average of triplicates ± S.D. with one-way analysis of variance significance p < 0.05*, p < 0.001***.

Article Snippet: Next, the blots were incubated overnight with 1:2,000 diluted primary mouse α-myc mAb (Cell Signaling, b118, 2% (w/v) BSA in TBST) at 4 °C, washing with TBST for 20 min (repeated 6 times), subsequently incubated with 1:10,000 diluted secondary rabbit α-mouse IRD680 (Cell Signaling, 2% (w/v) BSA in TBST) at room temperature, washed with TBST for 20 min (repeated 6 times), and finally, read-out occurred on an Odyssey Infrared Scanner (Li-Cor).

Techniques: Activity Assay, Plasmid Preparation, Purification, Labeling