Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Diverse resistance mechanisms to the third-generation ALK inhibitor lorlatinib in ALK-rearranged lung cancer

doi: 10.1158/1078-0432.CCR-19-1104

Figure Lengend Snippet: Clinical and molecular features of patients with tumor molecular profiling on biopsies obtained upon resistance to ALK inhibitors in the MATCH-R study.

Article Snippet: NF2 gene knock-out was performed with the CRISPR/Cas9 KO Plasmid (h) from Santa Cruz Biotechnology (sc-400504).

Techniques: Biomarker Discovery, Sequencing, RNA Sequencing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Diverse resistance mechanisms to the third-generation ALK inhibitor lorlatinib in ALK-rearranged lung cancer

doi: 10.1158/1078-0432.CCR-19-1104

Figure Lengend Snippet: NF2 loss of function mediates resistance to lorlatinib. A, Clinical course of patient MR135 and mutational profile of samples obtained on lorlatinib progression (PD, progressive Disease). B, Cell survival assay assessed with Cell Titer Glo of MR135 lorlatinib resistant cells from biopsy 1 (MR135-R1) treated for 7 days with the indicated concentrations of lorlatinib and vistusertib (AZD2014) alone or in combination. C, Immunoblot analysis from cell lysates of MR135-R1 treated for 24hs with the specified doses of lorlatinib, vistusertib (AZD2014) and ponatinib alone or in combination using indicated antibodies. D, Athymic nude mice bearing MR135-R2 PDX were administered lorlatinib or vistusertib 20 mg/kg orally. Tumor volumes, mean ±SD (n =8); (*** p < 0.001). E, Cell lysates from H3122 parental and H3122 cells with NF2 heterozygous deletions or homozygous deletions, generated by CRISPR-CAS9 gene editing, were immunoblotted to detect merlin expression. H3122 cells with bi-allelic NF2 knock-out lacked merlin expression. F, Cell survival assay of H3122 parental and H3122 NF2 knock-out (NF2 KO) cells treated with lorlatinib for 7 days. Cell survival was assessed by Cell Titer Glo. G, Cell proliferation assay of H3122 parental and H3122 NF2 KO cells untreated and treated with lorlatinib measured at baseline, day 2, day 5 and day 7. Cell viability was assessed with Cell Titer Glo. H, Caspase 3/7 activation (Caspase 3/7-Glo assay) relative to the number of live cells simultaneously assessed in the cell proliferation assay previously described. I, H3122 parental and NF2 KO cells were treated with the indicated doses of lorlatinib for 24hs. Cell lysates were immunoblotted to detect the selected proteins.

Article Snippet: NF2 gene knock-out was performed with the CRISPR/Cas9 KO Plasmid (h) from Santa Cruz Biotechnology (sc-400504).

Techniques: Clonogenic Cell Survival Assay, Western Blot, Generated, CRISPR, Expressing, Knock-Out, Proliferation Assay, Activation Assay, Glo Assay

Journal: Oncogene

Article Title: KIBRA exhibits MST-independent functional regulation of the Hippo signaling pathway in mammals.

doi: 10.1038/onc.2012.196

Figure Lengend Snippet: Figure 4. KIBRA expression antagonises YAP-induced transcriptional regulation. (a) mRNA expression of the YAP target genes BMP2, FGF1, RASSF4 and PDGFb was assessed by quantitative real-time PCR in MCF10A-YAP cells overexpressing KIBRA, Willin or Merlin. mRNA levels were compared with the empty vector control MCF10A-YAP cells. Willin and KIBRA overexpression increased BMP2 and RASSF4 mRNA levels and decreased FGF1 and PDGFb mRNA levels. Means were calculated from Ct values from two analyses conducted in triplicate. Error bars represent ±s.e. (n ¼ 6). MCF10A-YAP-vector vs MCF10A-YAP-KIBRA or MCF10A-YAP-Willin for all the analysed genes: *Po0.05, **Po0.01, ***Po0.001 Student’s t-test. MCF10A-YAP-vector vs MCF10A-YAP-Merlin: CTGF (P ¼ 0.129), FGF1 (P ¼ 0.005), BMP2 (P ¼ 0.585), RASSF4 (P ¼ 0.307), PDGFb (P ¼ 0.06), Student’s t-test. (b) Immunoblot analysis of KIBRA, Willin or Merlin overexpression in MCF10A-YAP cells, showing 2.7, 1.8 and 8.6-fold increases compared to empty vector control. b-actin was used as a loading control.

Article Snippet: All experiments were carried out at 80–90% confluency. pBABE Nf2 (Johnson et al.45) was obtained from Addgene (plasmid 14116).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Over Expression, Western Blot

Journal: Nature Communications

Article Title: Nf2-FAK signaling axis is critical for cranial bone ossification and regeneration

doi: 10.1038/s41467-025-57808-4

Figure Lengend Snippet: a Nf2 gene coding protein merlin was expressed in various tissues at embryonic 18.5 stage (upper panel, n = 3), as well as the embryonic 18.5 and postnatal (P3, P7, P14) skull tissues (lower panel, n = 3). b Nf2-cKO using Prx1-Cre resulted in severe birth defects in head shape and limbs, and skeletal staining ( c ) and micro-CT ( d ) showed Nf2-cKO led to serious deformities in frontal and parietal bones. The distance between two frontal bones ( c , red arrow) were significantly wide, and the distances ( c , black arrow) between nasal bone and coronal suture were notably enlarged in Nf2 mutant compared to the control ( n = 3) at E18.5 and P1. e 3D reconstruction from micro-CT images of cranial bones from E18.5 control ( n = 8) and Nf2-cKO ( n = 8). Isolated frontal bone ( h ) and parietal bone ( j ) from E18.5 control and Nf2-cKO mice. i , k Quantification of the size (length and width) of frontal bone ( i1 – 3 ) and parietal bone ( k1 – 3 ) of E18.5 control and Nf2-cKO ( n = 8). f , g Quantification of the volume, volume ratio, and surface defect area ratio of the frontal ( f1 – 3 ) and parietal bone ( g1 – 3 ) from E18.5 controls and Nf2-cKO ( n = 8). Scale bar=50 μm in ( b , c ) and 1 mm in ( d , e , h , j ). Data were expressed as means ± SD and each dot represents an individual biological replicate. P values were calculated by unpaired Student’s t-test with two-tailed analysis without adjustments. *** p < 0.001, ** p < 0.01, * p < 0.05. P-A: Posterior to Anterior. Definition of landmarks in frontal bone : 1 most anterior-superior point of the frontal bone; 2. most posterior-superior point of the frontal bone; 3. most posterior-lateral intersection of the frontal bone and parietal bone. Definition of landmarks in parietal bone : 1. most anterior-superior point of the frontal bone; 2. Most anterior-lateral point of the parietal bone; 3. most posterior-superior point of the frontal bone; 4. most posterior-lateral intersection of the parietal bone and interparietal bone. Abbreviation: Fb, frontal bone; Pb, parietal bone; iPb, interparietal bone; nb, nasal bone; mx, maxilla; md, mandible. Ob, occipital bone.

Article Snippet: Nf2 shRNA was purchased from Santa Cruz Biotechnology (sc-36053-SH, Santa Cruz, China), Nf2 cDNA was constructed by inserting the cDNA sequence into the pLentiPGK-puro vector using Gateway Cloning (Invitrogen).

Techniques: Staining, Micro-CT, Mutagenesis, Control, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Nf2-FAK signaling axis is critical for cranial bone ossification and regeneration

doi: 10.1038/s41467-025-57808-4

Figure Lengend Snippet: a Immunofluorescence images of EdU and Runx2 co-labeled osteoprogenitors at E12.5 coronal section of skull tissues. The percentage of Runx2 + cells ( b ), EdU + /Runx2 + ratio ( c ), EdU + /Runx2 - cells ( d ) were analyzed in the E12.5 controls and Nf2 mutants. e , f Nf2-cKO showed noticeably reduced extent of osteoblast activities and ossification using ALP staining at E12.5 and E17.5. g von Kossa staining showed remarkably reduced ossification in Nf2 mutants at the sections of E18.5 sagittal calvaria. Immunosignals of OPN ( h ), Runx2 ( i ) and Sp7 ( j ) were hardly found in Nf2 mutant sagittal calvaria at E18.5. In calvaria primary osteoblast culture system, ALP staining ( k ) and Alizarin S Red staining ( l ) showed severely impaired osteoblast differentiation and mineral matrix formation at D7 and D14, respectively. PCNA were significantly decreased at protein and gene expression levels in Nf2 mutant preosteoblasts ( m , o ). The activities of Runx2, Sp7 and collagen type ( I ) were significantly decreased in Nf2 mutant osteoblasts at D7 ( n ). Nf2-cKO osteoblast showed significantly declined levels of marker genes expressions ( ALP , Runx2 , Sp7 , OCN , Col1 , fibronectin, BSP, Dlx5, Msx2, Twist1/2 ) at D14 ( p ). Scale bar=50 μm in ( a , h – m ). scale bar=20 μm in ( e – j ). Data were expressed as means ± SD and each dot represents an individual biological replicate. P values were calculated by unpaired Student’s t-test with two-tailed analysis without adjustments. * p < 0.05 . ** p < 0.01, *** p < 0.001. abbreviation: Proliferating cell nuclear antigen (PCNA), Alkaline phosphatase (ALP), Osteopontin (OPN), Osteocalcin (OCN), bone sialoprotein (BSP), Type I collagen (Col1).

Article Snippet: Nf2 shRNA was purchased from Santa Cruz Biotechnology (sc-36053-SH, Santa Cruz, China), Nf2 cDNA was constructed by inserting the cDNA sequence into the pLentiPGK-puro vector using Gateway Cloning (Invitrogen).

Techniques: Immunofluorescence, Labeling, Staining, Mutagenesis, Gene Expression, Marker, Two Tailed Test

Journal: Nature Communications

Article Title: Nf2-FAK signaling axis is critical for cranial bone ossification and regeneration

doi: 10.1038/s41467-025-57808-4

Figure Lengend Snippet: a A scratch-wound assay was performed to examine MSC migration that isolated from Nf2 control and Nf2 mutant mice. The healing distances were calculated at each time point and analyzed ( n = 8) ( b ). c Primary osteoblasts were plating on fibronectin precoated plates with no fetal bovine serum for different time points, and the non-attached cells were washed, and the remaining osteoblasts were stained by crystal violet and counted for analysis, and Nf2 knockout led to significantly diminished cell adhesion. d The cultured adherent Nf2 mutant osteoblasts was significantly reduced after cell detachment experiment using 0.2% EDTA for 10 min. e F-actin staining by Phalloidin and β-tubulin staining showed that cytoskeletal assembly was disorganized in Nf2 mutant osteoblasts. f Rhotekin-RBD beads were added to lysate to pull down the RhoA, and Western Blotting were performed to measure the activity of RhoA in Nf2 mutants (BK030, Cytoskeleton, Inc.) and the data showed that RhoA activity was significantly decreased in Nf2 mutant MSC. Scale bar= 50 μm in ( a , e ). Data were expressed as means ± SD and each dot represents an individual biological replicate. P values were calculated by unpaired Student’s t-test with two-tailed analysis without adjustments. * p < 0.05 . ** p < 0.01, *** p < 0.001.

Article Snippet: Nf2 shRNA was purchased from Santa Cruz Biotechnology (sc-36053-SH, Santa Cruz, China), Nf2 cDNA was constructed by inserting the cDNA sequence into the pLentiPGK-puro vector using Gateway Cloning (Invitrogen).

Techniques: Scratch Wound Assay Assay, Migration, Isolation, Control, Mutagenesis, Staining, Knock-Out, Cell Culture, Western Blot, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Nf2-FAK signaling axis is critical for cranial bone ossification and regeneration

doi: 10.1038/s41467-025-57808-4

Figure Lengend Snippet: a Confocal images of immunostaining assay showed that the extent of p-Paxillin-labeled focal adhesion and Phalloidin-labeled F-actin in Nf2 mutant MSC and control. b arrow indicated higher magnification of FA in ( a ). c The area of FA size was quantified using ImageJ ( n = 12). d Live cell imaging analysis from transient transfection of Paxillin-GFP into primary osteoblasts showed that Paxillin-GFP -labeled focal adhesion dynamics were properly growing in control osteoblasts (enlarged magnification shown in d’ in arrow), but Paxillin-GFP -labeled focal adhesions were mainly situated in cytoplasm in Nf2 mutant osteoblasts (enlarged magnification shown in d” in arrow). e The ratio between properly developed Paxillin + cells and total cells in Nf2 mutants was notably lower compared to the controls ( n = 3). Scale bar: 10 μm in ( a – c ), 50 μm in ( d ) . Data were expressed as means ± SD and each dot represents an individual biological replicate. P values were calculated by unpaired Student’s t-test with two-tailed analysis without adjustments. * p < 0.05 . ** p < 0.01, *** p < 0.001.

Article Snippet: Nf2 shRNA was purchased from Santa Cruz Biotechnology (sc-36053-SH, Santa Cruz, China), Nf2 cDNA was constructed by inserting the cDNA sequence into the pLentiPGK-puro vector using Gateway Cloning (Invitrogen).

Techniques: Immunostaining, Labeling, Mutagenesis, Control, Live Cell Imaging, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: Nf2-FAK signaling axis is critical for cranial bone ossification and regeneration

doi: 10.1038/s41467-025-57808-4

Figure Lengend Snippet: a The Nf2 knockout primary osteoblasts (OB) at differentiation stage of D7 showed decreased activity of phospho-397FAK. b ALP staining exhibited that overexpression of Nf2 or FAK using lentivirus mediating system can sufficiently rescue the defective activities of Nf2 mutant osteoblasts. c The levels of phospho-397FAK, Runx2, Sp7 and collagen type I were sufficiently rescued after the lentivirus mediated Nf2 overexpression in Nf2 mutant osteoblasts. d co-IP assay showed that FLAG-FAK and HA-NF2 forms an interaction in 293 T. e co-IP assay confirms that Nf2 and FAK interacted in primary osteoblasts. f , g NanoBit protein:protein interaction system (N2015, Promega) using paired Nf2 and FAK plasmids based on structural conformation ( f ) showed that C-terminal domain of Nf2 protein can physically interact with the N-terminal domain of FAK in osteoblast cell line MC3T3-E1(3T3)( g ). g The bioluminescence ratios between NF2-FAK interaction and negative control ( n = 3) were analyzed using Nano-Glo® Live-cell Reagent (N1661, Promega) (Y: bioluminescence value; X: paired plasmids, such as N represents NF2; F represents FAK; C represents C terminal; N represents N terminal; 1.1 represents LgBiT, 2.1 represents SmBiT). h The immunosignals of phospho-FAK (397) were largely diminished in Runx2 + osteoprogenitor in Nf2 mutants at E12.5 coronal section of the skull (arrow). i The immunosignals of phospho-FAK (397) were significantly reduced in Nf2 mutants at E18.5 sagittal section of the skull. Scale bar: 20 μm in ( b ) 50 μm in ( h , i ). Data were expressed as means ± SD and each dot represents an individual biological replicate. P values were calculated by unpaired Student’s t-test with two-tailed analysis without adjustments. * p < 0.05 . ** p < 0.01, *** p < 0.001.

Article Snippet: Nf2 shRNA was purchased from Santa Cruz Biotechnology (sc-36053-SH, Santa Cruz, China), Nf2 cDNA was constructed by inserting the cDNA sequence into the pLentiPGK-puro vector using Gateway Cloning (Invitrogen).

Techniques: Knock-Out, Activity Assay, Staining, Over Expression, Mutagenesis, Co-Immunoprecipitation Assay, Negative Control, Two Tailed Test

Journal: Nature Communications

Article Title: Nf2-FAK signaling axis is critical for cranial bone ossification and regeneration

doi: 10.1038/s41467-025-57808-4

Figure Lengend Snippet: a The activities of phospho-PLC-γ and phospho-Akt were particularly reduced in Nf2 mutant osteoblasts. b The activity of phospho-Erk1/2 was specially decreased, and while the activities of phospho-p38 and phospho-JNK were not altered in Nf2 mutant osteoblasts ( a – b ). c – e In Nf2 mutant primary osteoblasts, coIP assay showed that Nf2 deletion led to decreased connection between FAK and PI3K catalytic subunit p110. f , g The immunosignals of phospho-Akt and phospho-Erk1/2 at sagittal section of E18.5 skull tissue were notably reduced in Nf2 mutants (arrow). h , i The immunosignals of phospho-Akt and phospho-Erk1/2 in Nf2 mutants at the section of E12.5 cranial primordium were remarkably decreased in Runx2 + osteoprogenitors (arrow). Scale bar: 50 μm in ( h ), 100 μm in ( f , i ). Data were expressed as means ± SD and each dot represents an individual biological replicate. P values were calculated by unpaired Student’s t-test with two-tailed analysis without adjustments. OB, primary osteoblasts.

Article Snippet: Nf2 shRNA was purchased from Santa Cruz Biotechnology (sc-36053-SH, Santa Cruz, China), Nf2 cDNA was constructed by inserting the cDNA sequence into the pLentiPGK-puro vector using Gateway Cloning (Invitrogen).

Techniques: Mutagenesis, Activity Assay, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Nf2-FAK signaling axis is critical for cranial bone ossification and regeneration

doi: 10.1038/s41467-025-57808-4

Figure Lengend Snippet: a The inhibition of the FAK activity using inhibitor PF573228 resulted in significantly decreased activities of phospho-Erk1/2 and phospho-Akt and reduced activities of Runx2 and Sp7 in primary osteoblasts for 24 h. b The pharmaceutical activation of FAK on phospho-FAK (397) using Adhesamine (50 μM) can sufficiently rescue the activities of phospho-397FAK and its downstream signaling phospho-Erk1/2 and phospho-Akt in Nf2 mutant osteoblasts for 24 h. c Adding Akt activator, Erk1/2 activator, and FAK activator in primary osteoblasts for 14 days can sufficiently rescue deficient ossification in Nf2 mutant osteoblasts, and solvents used for the activators applied to the control groups. Data were expressed as means ± SD with at least three biological replicates. Scale bar: 50 μm in ( c ). OB, primary osteoblasts.

Article Snippet: Nf2 shRNA was purchased from Santa Cruz Biotechnology (sc-36053-SH, Santa Cruz, China), Nf2 cDNA was constructed by inserting the cDNA sequence into the pLentiPGK-puro vector using Gateway Cloning (Invitrogen).

Techniques: Inhibition, Activity Assay, Activation Assay, Mutagenesis, Control

Journal: Nature Communications

Article Title: Nf2-FAK signaling axis is critical for cranial bone ossification and regeneration

doi: 10.1038/s41467-025-57808-4

Figure Lengend Snippet: a Cranial defects were imaged at different stage of frontal and parietal bone (D0, D7, D14, D21) in Nf2 mutant mice (2 m, n = 3 at each stage) using micro-CT , as it indicated in the experimental design in ( b ). c Nf2 mutants showed delayed healing of cranial defects at different stage of post injury (D7, D14, D21). d Migrated suture stem cells/progeny from the control and Nf2 mutant mice (2 m, n = 3) were traced using R26-tdTomato , and EdU was incorporated into the mice to evaluate the proliferation of migrated cells at post-injury 10 days at injury site. Nf2 mutant frontal and parietal bone showed less migrated suture stem cells/progeny and few EdU + / R26-tdTomato + can be observed ( d , e , arrow, n = 3) compared to widely distributed EdU + / R26-tdTomato + cells at frontal and parietal injury site in the controls. f The whole-mount images of migrating R26-tdTomato + stem cells/progeny towards (arrow) and at injury sites (dotted line) at post-injury D7 upon Nf2 deficiency in Prx1 + suture stem cells from control and Nf2 mutant mice (2 m, n = 3). g Mechanism of Nf2 mediated FAK signaling axis in cranial bone development and regeneration. Nf2 acts as a regulator to modulate the activity of phospho-397FAK and physically interacts with FAK. Deletion of Nf2 leads to defective FA assembly, cytoskeletal organization to influence MSC migration. Nf2-FAK especially mediated cellular Erk1/2 and p110-PI3K/Akt signaling to regulate MSC migration and differentiation. Scale bar: 50 μm in ( a , d , f ). Data were expressed as means ± SD and each dot represents an individual biological replicate. P values were calculated by unpaired Student’s t-test with two-tailed analysis without adjustments. * p < 0.05 . ** p < 0.01, *** p < 0.001. Fb, frontal bone; Pb, parietal bone.

Article Snippet: Nf2 shRNA was purchased from Santa Cruz Biotechnology (sc-36053-SH, Santa Cruz, China), Nf2 cDNA was constructed by inserting the cDNA sequence into the pLentiPGK-puro vector using Gateway Cloning (Invitrogen).

Techniques: Mutagenesis, Micro-CT, Control, Activity Assay, Migration, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Missense mutations in the NF2 gene result in the quantitative loss of merlin protein and minimally affect protein intrinsic function.

doi: 10.1073/pnas.1102198108

Figure Lengend Snippet: Fig. 1. Quantification of merlin protein and RNA expression. (A) Western blot of merlin proteins from various microdissected NF2-associated tumor samples. (B) Immunofluorescence staining for merlin (red) in tumor speci- mens and normal tissue derived from patients with NF2. The nuclei of cells are counterstained with Hoechst (blue). (C) RT-PCR for NF2 mRNA expression from various NF2-associated tumors.

Article Snippet: Mutations in the NF2 gene were generated in pCMV6-Entry vectors with the full-length wild-type NF2 gene (Origene).

Techniques: RNA Expression, Western Blot, Staining, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Missense mutations in the NF2 gene result in the quantitative loss of merlin protein and minimally affect protein intrinsic function.

doi: 10.1073/pnas.1102198108

Figure Lengend Snippet: Fig. 2. Half-life kinetic analysis of reduction of mutant merlin proteins. (A) Radioactive 35S pulse chase assay of wild-type merlin and different merlin missense mutants at 0, 6, and 24 h demonstrating increased turnover and degradation of mutant merlin compared with wild-type control. (B) Radioactive 35S pulse chase assay at several time points (in hours) demonstrating the degradation kinetics of merlin mutant K413E compared with wild-type control. (C) Liquid scintillation measurement of 35S-labeled merlin demonstrating an increased degradation rate of K413E merlin mutant compared with wild-type control.

Article Snippet: Mutations in the NF2 gene were generated in pCMV6-Entry vectors with the full-length wild-type NF2 gene (Origene).

Techniques: Mutagenesis, Pulse Chase, Control, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Missense mutations in the NF2 gene result in the quantitative loss of merlin protein and minimally affect protein intrinsic function.

doi: 10.1073/pnas.1102198108

Figure Lengend Snippet: Fig. 3. Mutant merlin protein maintains intrinsic functional capacity. (A) Immunofluorescence staining for F-actin (green) and merlin mutants (red) in CH157 NM cells transfected with wild-type merlin, merlin mutants, as well as empty vector (blank). The nuclei of cells were counterstained with Hoechst (blue). Arrows point to F-actin stress fibers and arrowheads point to mutant merlin. (B) Western blot of lysates from CH157 NM cells transfected with either wild-type or K413E mutant merlin protein. K413E-transfected cells demonstrate increased merlin ubiquitylation and up-regulation of β-catenin and cyclin D1 cytoplasmic expression. (C) MTT proliferation assay demon- strating suppression of CH157 NM cell growth with mutant merlin trans- fection compared with empty vector (blank). (D) Flow cytometric BrdU-PI cell cycle analysis demonstrating reduced proliferation in CH157 NM cells transfected with L46R and A211D merlin mutants compared with wild type and empty vector (blank).

Article Snippet: Mutations in the NF2 gene were generated in pCMV6-Entry vectors with the full-length wild-type NF2 gene (Origene).

Techniques: Mutagenesis, Functional Assay, Staining, Transfection, Plasmid Preparation, Western Blot, Expressing, Proliferation Assay, Cell Cycle Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Missense mutations in the NF2 gene result in the quantitative loss of merlin protein and minimally affect protein intrinsic function.

doi: 10.1073/pnas.1102198108

Figure Lengend Snippet: Fig. 4. Increased cellular expression levels of mutant merlin protein after treatment with proteostasis regulators. (A) Radioactive 35S pulse chase assay demonstrating prolonged K413E merlin mutant protein half-life with celastrol and quercetin treatment. (B) 35S liquid scintillation measurement demon- strating a quantitative decrease in K413E mutant merlin degradation rate with celastrol and high dose quercetin treatment. (C) Radioactive 35S pulse chase assay at several time points (in hours) demonstrating the degradation kinetics of merlin mutant K413E before and after treatment with celastrol compared with wild type. (D) 35S liquid scintillation measurement at several time points (in hours) demonstrating the degradation kinetics of mutant merlin K413E before and after treatment with celastrol compared with wild-type control. (E) Western blot of lysates from CH157 NM cells transfected with merlin mutant K413E before and after celastrol treatment, demonstrating decreased ubiquitin binding, increased Hsp70 and TCP1 binding to immunoprecipitated merlin protein, as well as decreased β-catenin expression after celastrol treatment. (F) Flow cytometric analysis of CH157 NM cells transfected with K413 mutant merlin demonstrating decreased cell proliferation after treatment with celastrol. (G) Immunofluorescence staining for F-actin (green) and merlin K413E mutant protein (red) in CH157 NM cells before and after celastrol treatment. The nuclei of cells are counterstained with Hoechst (blue).

Article Snippet: Mutations in the NF2 gene were generated in pCMV6-Entry vectors with the full-length wild-type NF2 gene (Origene).

Techniques: Expressing, Mutagenesis, Pulse Chase, Control, Western Blot, Transfection, Ubiquitin Proteomics, Binding Assay, Immunoprecipitation, Staining