Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 1. Flow cytometry sorting of the combinatorial Scl library. (A) Library expression was monitored by staining with a phycoerythrin- conjugated antibody binding to a primary anti-c-Myc antibody. The purple rectangle gate indicates cells with the highest expression. (B) The library was incubated with 5 nM LRP4, and the low-affinity library fraction was collected (purple triangle). (C) The low-affinity library was incubated with 650 nM LRP6, and the purple rectangle represents clones that bind to LRP6.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Flow Cytometry, Expressing, Staining, Binding Assay, Incubation, Clone Assay

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 2. Identification of affinity-reducing mutations. Heat maps demonstrating significantly enriched Scl variants in (A) LRP4LOW library compared to SclNAIVE library fractions; (B) LRP4LOWLRP6 library compared to LRP4LOW library fractions; (C) LRP4LOW library compared to SclNAIVE library fractions that overlap with the LRP4LOWLRP6 library. The heat maps present the log2 transformation of the ER (red scale bar on the right-hand side) and highlight single mutations that significantly (A) reduce the binding affinity to LRP4, (B) reduce the binding affinity to LRP4 and retain binding to LRP6, and (C) overlap in (A) and (B). The substituting amino acids are shown on the X-axis, and the substituted positions are shown on the Y-axis. Statistical significance was determined by a two-sided Poisson exact test and multi-test corrected by the Benjamini–Hochberg FDR.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Transformation Assay, Binding Assay

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 4. YSD binding of SclWT and the selected single-mutation variants to LRP4. Geometric mean fluorescence intensity (Geo MFI) is presented as a fold change. Recombinant yeast cells expressing SclWT or its variants were incubated with (A) 1 nM, (B) 10 nM, or (C) 50 nM soluble LRP4. The binding signal of each Scl variant was normalized first to the expression signal of the corresponding variant and then to the binding signal of SclWT at the respective LRP4 concentration. Each experiment was repeated at least three times, and the results are presented as means SD. Statistical significance was assessed using an unpaired, two-tailed Student’s t-test. *P ≤0.05; **P ≤0.01; ***P ≤0.001.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Binding Assay, Mutagenesis, Recombinant, Expressing, Incubation, Variant Assay, Concentration Assay, Two Tailed Test

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 7. SPR analysis of binding of purified SclWT and Scl single-mutation variants to LRP4. SPR data showing binding of (A) SclWT, (B) SclK75Q, (C) SclK75E, and (D) SclV136D to 3 μg of immobilized LRP4 receptor. Different protein concentrations are represented by different colors.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Binding Assay, Mutagenesis

Journal: Experimental Physiology

Article Title: Acute high‐intensity muscle contraction moderates AChR gene expression independent of rapamycin‐sensitive mTORC1 pathway in rat skeletal muscle

doi: 10.1113/EP091006

Figure Lengend Snippet: Primer sequences for RT‐PCR.

Article Snippet: After blocking, membranes were washed and incubated with primary antibodies against agrin (cat. no. MAB5204; Merck, Darmstadt, Germany), LRP4 (cat. no. MAB5948; R&D Systems, Minneapolis, MN, USA), Dok‐7 (cat. no. sc390856; Santa Cruz Biotechnology, Dallas, TX, USA), phosphorylated‐MuSK (cat. no. ab192583; Abcam, Toronto, ON, Canada), MuSK (cat. no. sc134398; Santa Cruz Biotechnology), rapsyn (cat. no. ab156002; Abcam), phospho‐p70S6K (Thr389, cat. no. 9205), p70S6K (cat. no. 9202), phospho‐rpS6 (Ser240/244, cat. no. 2215), rpS6 (cat. no. 2317), phospho‐ULK1 (Ser317, cat. no. 12753; Thr757, cat. no.14202), ULK1 (cat. no. 8054), LC3 (cat. no. 2775), phospho‐Akt (Ser473, cat. no. 9271; Thr308, cat. no. 13038), Akt (cat. no. 9272), HDAC4 (cat. no. 2072), histone H3 (cat. no. 9715; Cell Signalling Technology, Danvers, MA, USA) and p62 (cat. no. PM045; Medical & Biological Laboratories, Aichi, Japan).

Techniques:

Journal: Experimental Physiology

Article Title: Acute high‐intensity muscle contraction moderates AChR gene expression independent of rapamycin‐sensitive mTORC1 pathway in rat skeletal muscle

doi: 10.1113/EP091006

Figure Lengend Snippet: mRNA expression of NMJ‐related factors in the early recovery phase from an acute bout of muscle contraction. (a) Agrn , (b) Lrp4 , (c) Musk , (d) Dok7 , (e) Chrna1 , (f) Chrnb1 , (g) Chrnd , (h) Rapsn and (i) Chrne . Mean values are shown with individual values in bar and dot plots. * P < 0.05 versus CON. AU, arbitrary units; CON, control.

Article Snippet: After blocking, membranes were washed and incubated with primary antibodies against agrin (cat. no. MAB5204; Merck, Darmstadt, Germany), LRP4 (cat. no. MAB5948; R&D Systems, Minneapolis, MN, USA), Dok‐7 (cat. no. sc390856; Santa Cruz Biotechnology, Dallas, TX, USA), phosphorylated‐MuSK (cat. no. ab192583; Abcam, Toronto, ON, Canada), MuSK (cat. no. sc134398; Santa Cruz Biotechnology), rapsyn (cat. no. ab156002; Abcam), phospho‐p70S6K (Thr389, cat. no. 9205), p70S6K (cat. no. 9202), phospho‐rpS6 (Ser240/244, cat. no. 2215), rpS6 (cat. no. 2317), phospho‐ULK1 (Ser317, cat. no. 12753; Thr757, cat. no.14202), ULK1 (cat. no. 8054), LC3 (cat. no. 2775), phospho‐Akt (Ser473, cat. no. 9271; Thr308, cat. no. 13038), Akt (cat. no. 9272), HDAC4 (cat. no. 2072), histone H3 (cat. no. 9715; Cell Signalling Technology, Danvers, MA, USA) and p62 (cat. no. PM045; Medical & Biological Laboratories, Aichi, Japan).

Techniques: Expressing, Control

Journal: Experimental Physiology

Article Title: Acute high‐intensity muscle contraction moderates AChR gene expression independent of rapamycin‐sensitive mTORC1 pathway in rat skeletal muscle

doi: 10.1113/EP091006

Figure Lengend Snippet: Protein expression of NMJ‐related factors in the early recovery phase from an acute bout of muscle contraction. (a) Representative images, (b) agrin, (c) Lrp4, (d) phosphorylated MuSK at Tyr755, (e) MuSK, (f) Dok‐7 and (g) rapsyn. Mean values are shown with individual values in bar and dot plots. * P < 0.05 versus CON. AU, arbitrary units; CON, control.

Article Snippet: After blocking, membranes were washed and incubated with primary antibodies against agrin (cat. no. MAB5204; Merck, Darmstadt, Germany), LRP4 (cat. no. MAB5948; R&D Systems, Minneapolis, MN, USA), Dok‐7 (cat. no. sc390856; Santa Cruz Biotechnology, Dallas, TX, USA), phosphorylated‐MuSK (cat. no. ab192583; Abcam, Toronto, ON, Canada), MuSK (cat. no. sc134398; Santa Cruz Biotechnology), rapsyn (cat. no. ab156002; Abcam), phospho‐p70S6K (Thr389, cat. no. 9205), p70S6K (cat. no. 9202), phospho‐rpS6 (Ser240/244, cat. no. 2215), rpS6 (cat. no. 2317), phospho‐ULK1 (Ser317, cat. no. 12753; Thr757, cat. no.14202), ULK1 (cat. no. 8054), LC3 (cat. no. 2775), phospho‐Akt (Ser473, cat. no. 9271; Thr308, cat. no. 13038), Akt (cat. no. 9272), HDAC4 (cat. no. 2072), histone H3 (cat. no. 9715; Cell Signalling Technology, Danvers, MA, USA) and p62 (cat. no. PM045; Medical & Biological Laboratories, Aichi, Japan).

Techniques: Expressing, Control

Journal: Experimental Physiology

Article Title: Acute high‐intensity muscle contraction moderates AChR gene expression independent of rapamycin‐sensitive mTORC1 pathway in rat skeletal muscle

doi: 10.1113/EP091006

Figure Lengend Snippet: mRNA expression of NMJ‐related factors in the late recovery phase from an acute bout of muscle contraction. (a) Agrn , (b) Lrp4 , (c) Musk , (d) Dok7 , (e) Chrna1 , (f) Chrnb1 , (g) Chrnd , (h) Rapsn and (i) Chrne . Mean values are shown with individual values in bar and dot plots. * P < 0.05 versus CON. AU, arbitrary units; CON, control.

Article Snippet: After blocking, membranes were washed and incubated with primary antibodies against agrin (cat. no. MAB5204; Merck, Darmstadt, Germany), LRP4 (cat. no. MAB5948; R&D Systems, Minneapolis, MN, USA), Dok‐7 (cat. no. sc390856; Santa Cruz Biotechnology, Dallas, TX, USA), phosphorylated‐MuSK (cat. no. ab192583; Abcam, Toronto, ON, Canada), MuSK (cat. no. sc134398; Santa Cruz Biotechnology), rapsyn (cat. no. ab156002; Abcam), phospho‐p70S6K (Thr389, cat. no. 9205), p70S6K (cat. no. 9202), phospho‐rpS6 (Ser240/244, cat. no. 2215), rpS6 (cat. no. 2317), phospho‐ULK1 (Ser317, cat. no. 12753; Thr757, cat. no.14202), ULK1 (cat. no. 8054), LC3 (cat. no. 2775), phospho‐Akt (Ser473, cat. no. 9271; Thr308, cat. no. 13038), Akt (cat. no. 9272), HDAC4 (cat. no. 2072), histone H3 (cat. no. 9715; Cell Signalling Technology, Danvers, MA, USA) and p62 (cat. no. PM045; Medical & Biological Laboratories, Aichi, Japan).

Techniques: Expressing, Control

Journal: Experimental Physiology

Article Title: Acute high‐intensity muscle contraction moderates AChR gene expression independent of rapamycin‐sensitive mTORC1 pathway in rat skeletal muscle

doi: 10.1113/EP091006

Figure Lengend Snippet: Protein expression of NMJ‐related factors in the late recovery phase from an acute bout of muscle contraction. (a) Representative images, (b) agrin, (c) Lrp4, (d) phosphorylated MuSK at Tyr755, (e) MuSK, (f) Dok‐7 and (g) rapsyn. Mean values are shown with individual values in bar and dot plots. * P < 0.05 versus CON. AU, arbitrary units; CON, control.

Article Snippet: After blocking, membranes were washed and incubated with primary antibodies against agrin (cat. no. MAB5204; Merck, Darmstadt, Germany), LRP4 (cat. no. MAB5948; R&D Systems, Minneapolis, MN, USA), Dok‐7 (cat. no. sc390856; Santa Cruz Biotechnology, Dallas, TX, USA), phosphorylated‐MuSK (cat. no. ab192583; Abcam, Toronto, ON, Canada), MuSK (cat. no. sc134398; Santa Cruz Biotechnology), rapsyn (cat. no. ab156002; Abcam), phospho‐p70S6K (Thr389, cat. no. 9205), p70S6K (cat. no. 9202), phospho‐rpS6 (Ser240/244, cat. no. 2215), rpS6 (cat. no. 2317), phospho‐ULK1 (Ser317, cat. no. 12753; Thr757, cat. no.14202), ULK1 (cat. no. 8054), LC3 (cat. no. 2775), phospho‐Akt (Ser473, cat. no. 9271; Thr308, cat. no. 13038), Akt (cat. no. 9272), HDAC4 (cat. no. 2072), histone H3 (cat. no. 9715; Cell Signalling Technology, Danvers, MA, USA) and p62 (cat. no. PM045; Medical & Biological Laboratories, Aichi, Japan).

Techniques: Expressing, Control