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lis1 pafah1b1 rabbit polyclonal antibody  (Cell Signaling Technology Inc)
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Lis1 Pafah1b1 Rabbit Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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lis1 rabbit polyclonal 484/485 antibody  (Santa Cruz Biotechnology)
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<t>Lis1</t> KO impacts axonal function in adult mouse DRG neurons. A , Cultured DRG neurons from no Cre control exposed to 4-OH tamoxifen for 5 d expressed only tdTomato (red) showing no signs of recombination. B , In contrast, Lis1 KO neurons had strong GFP expression (green) demonstrating recombination. C , 4-OH tamoxifen reduced Lis1 protein levels in Lis1 KO neurons relative to no Cre control neurons (CON). D , Intraperitoneal injection of 2 × 8 mg tamoxifen in Lis1 KO mice resulted in GFP expression in intact DRGs after 4 d. Arrows point to DRG plasma membranes. E , Cultured DRG neurons prepared from intraperitoneally injected, no Cre control animals expressed only tdTomato (red). NF (blue) was prominent along axon shafts (white arrow) but less prominent in axon terminals (arrowhead). F , DRG neurons prepared from intraperitoneally injected Lis1 KO mice continued to express GFP (green) in culture, and NF (blue) was most prominent in distal axons and enriched in in varicosities (arrow). G , Insets from E , F have been digitally enlarged to show axonal varicosities (arrows). The bar graph in G shows the average number of varicosities per 100 µm of axon from N = 3 CON (45 mm total axon length) and three Lis1 KO (27 mm total axon length) mice. H , Kymographs were generated from time-lapse movies of LysoTracker labeled organelles in GFP-positive axons. The bar graph shows the percentage moving retrogradely in Lis1 KO and no flLis1 control cultures (CON). A total of 27 100 µm axon segments were analyzed from N = 2 CON and N = 2 Lis1 KO mice. A total of n = 521 control and n = 699 KO organelles were analyzed. I , Cultured DRG neurons prepared from intraperitoneally injected, no flLis1 control and Lis1 KO mice were immunostained with neuron-specific antibodies, and the percentage of neurons with growing axons was determined from N = 4 CON and N = 5 Lis1 KO mice. A total of n = 2219 control neurons and n = 2410 Lis1 KO neurons were analyzed. Bars in G-I indicate mean ± SD. Significance determined by Mann–Whitney test ( G ), Student’s t test ( H , I ), * p < 0.05, *** p < 0.001 (see for details). Scale bars: 20 µm ( A , D , E ), 5 µm ( B ), and 50 µm ( I ).
Lis1 Rabbit Polyclonal 484/485 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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lis1 rabbit polyclonal 484/485 antibody - by Bioz Stars, 2026-10
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lis1 rabbit polyclonal  (Santa Cruz Biotechnology)
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<t>Lis1</t> KO impacts axonal function in adult mouse DRG neurons. A , Cultured DRG neurons from no Cre control exposed to 4-OH tamoxifen for 5 d expressed only tdTomato (red) showing no signs of recombination. B , In contrast, Lis1 KO neurons had strong GFP expression (green) demonstrating recombination. C , 4-OH tamoxifen reduced Lis1 protein levels in Lis1 KO neurons relative to no Cre control neurons (CON). D , Intraperitoneal injection of 2 × 8 mg tamoxifen in Lis1 KO mice resulted in GFP expression in intact DRGs after 4 d. Arrows point to DRG plasma membranes. E , Cultured DRG neurons prepared from intraperitoneally injected, no Cre control animals expressed only tdTomato (red). NF (blue) was prominent along axon shafts (white arrow) but less prominent in axon terminals (arrowhead). F , DRG neurons prepared from intraperitoneally injected Lis1 KO mice continued to express GFP (green) in culture, and NF (blue) was most prominent in distal axons and enriched in in varicosities (arrow). G , Insets from E , F have been digitally enlarged to show axonal varicosities (arrows). The bar graph in G shows the average number of varicosities per 100 µm of axon from N = 3 CON (45 mm total axon length) and three Lis1 KO (27 mm total axon length) mice. H , Kymographs were generated from time-lapse movies of LysoTracker labeled organelles in GFP-positive axons. The bar graph shows the percentage moving retrogradely in Lis1 KO and no flLis1 control cultures (CON). A total of 27 100 µm axon segments were analyzed from N = 2 CON and N = 2 Lis1 KO mice. A total of n = 521 control and n = 699 KO organelles were analyzed. I , Cultured DRG neurons prepared from intraperitoneally injected, no flLis1 control and Lis1 KO mice were immunostained with neuron-specific antibodies, and the percentage of neurons with growing axons was determined from N = 4 CON and N = 5 Lis1 KO mice. A total of n = 2219 control neurons and n = 2410 Lis1 KO neurons were analyzed. Bars in G-I indicate mean ± SD. Significance determined by Mann–Whitney test ( G ), Student’s t test ( H , I ), * p < 0.05, *** p < 0.001 (see for details). Scale bars: 20 µm ( A , D , E ), 5 µm ( B ), and 50 µm ( I ).
Lis1 Rabbit Polyclonal, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit polyclonal lis1  (Santa Cruz Biotechnology)
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<t>NAGK-Lis1</t> interaction during the cell cycle. PLA was carried out on fixed HEK293T cells using primary antibodies against NAGK and Lis1, subsequent ICC was performed using anti-tubulin antibody and then DAPI staining was done. In interphase (A) and prophase cells (B), NAGK-Lis1 interactions were found on nuclear membranes (arrowheads). In metaphase (C), anaphase (D), telophase (E) and cytokinesis (F) stages, PLA signals were observed on chromosomes and/or on MTs (red arrowheads), and in other cytoplasmic areas (white arrowheads). Scale bar; 5 μm.
Rabbit Polyclonal Lis1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit polyclonal anti lis1  (Bethyl)
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<t>NAGK-Lis1</t> interaction during the cell cycle. PLA was carried out on fixed HEK293T cells using primary antibodies against NAGK and Lis1, subsequent ICC was performed using anti-tubulin antibody and then DAPI staining was done. In interphase (A) and prophase cells (B), NAGK-Lis1 interactions were found on nuclear membranes (arrowheads). In metaphase (C), anaphase (D), telophase (E) and cytokinesis (F) stages, PLA signals were observed on chromosomes and/or on MTs (red arrowheads), and in other cytoplasmic areas (white arrowheads). Scale bar; 5 μm.
Rabbit Polyclonal Anti Lis1, supplied by Bethyl, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit polyclonal anti lis1  (Danaher Inc)
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<t>NAGK-Lis1</t> interaction during the cell cycle. PLA was carried out on fixed HEK293T cells using primary antibodies against NAGK and Lis1, subsequent ICC was performed using anti-tubulin antibody and then DAPI staining was done. In interphase (A) and prophase cells (B), NAGK-Lis1 interactions were found on nuclear membranes (arrowheads). In metaphase (C), anaphase (D), telophase (E) and cytokinesis (F) stages, PLA signals were observed on chromosomes and/or on MTs (red arrowheads), and in other cytoplasmic areas (white arrowheads). Scale bar; 5 μm.
Rabbit Polyclonal Anti Lis1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Lis1 KO impacts axonal function in adult mouse DRG neurons. A , Cultured DRG neurons from no Cre control exposed to 4-OH tamoxifen for 5 d expressed only tdTomato (red) showing no signs of recombination. B , In contrast, Lis1 KO neurons had strong GFP expression (green) demonstrating recombination. C , 4-OH tamoxifen reduced Lis1 protein levels in Lis1 KO neurons relative to no Cre control neurons (CON). D , Intraperitoneal injection of 2 × 8 mg tamoxifen in Lis1 KO mice resulted in GFP expression in intact DRGs after 4 d. Arrows point to DRG plasma membranes. E , Cultured DRG neurons prepared from intraperitoneally injected, no Cre control animals expressed only tdTomato (red). NF (blue) was prominent along axon shafts (white arrow) but less prominent in axon terminals (arrowhead). F , DRG neurons prepared from intraperitoneally injected Lis1 KO mice continued to express GFP (green) in culture, and NF (blue) was most prominent in distal axons and enriched in in varicosities (arrow). G , Insets from E , F have been digitally enlarged to show axonal varicosities (arrows). The bar graph in G shows the average number of varicosities per 100 µm of axon from N = 3 CON (45 mm total axon length) and three Lis1 KO (27 mm total axon length) mice. H , Kymographs were generated from time-lapse movies of LysoTracker labeled organelles in GFP-positive axons. The bar graph shows the percentage moving retrogradely in Lis1 KO and no flLis1 control cultures (CON). A total of 27 100 µm axon segments were analyzed from N = 2 CON and N = 2 Lis1 KO mice. A total of n = 521 control and n = 699 KO organelles were analyzed. I , Cultured DRG neurons prepared from intraperitoneally injected, no flLis1 control and Lis1 KO mice were immunostained with neuron-specific antibodies, and the percentage of neurons with growing axons was determined from N = 4 CON and N = 5 Lis1 KO mice. A total of n = 2219 control neurons and n = 2410 Lis1 KO neurons were analyzed. Bars in G-I indicate mean ± SD. Significance determined by Mann–Whitney test ( G ), Student’s t test ( H , I ), * p < 0.05, *** p < 0.001 (see for details). Scale bars: 20 µm ( A , D , E ), 5 µm ( B ), and 50 µm ( I ).

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Lis1 KO impacts axonal function in adult mouse DRG neurons. A , Cultured DRG neurons from no Cre control exposed to 4-OH tamoxifen for 5 d expressed only tdTomato (red) showing no signs of recombination. B , In contrast, Lis1 KO neurons had strong GFP expression (green) demonstrating recombination. C , 4-OH tamoxifen reduced Lis1 protein levels in Lis1 KO neurons relative to no Cre control neurons (CON). D , Intraperitoneal injection of 2 × 8 mg tamoxifen in Lis1 KO mice resulted in GFP expression in intact DRGs after 4 d. Arrows point to DRG plasma membranes. E , Cultured DRG neurons prepared from intraperitoneally injected, no Cre control animals expressed only tdTomato (red). NF (blue) was prominent along axon shafts (white arrow) but less prominent in axon terminals (arrowhead). F , DRG neurons prepared from intraperitoneally injected Lis1 KO mice continued to express GFP (green) in culture, and NF (blue) was most prominent in distal axons and enriched in in varicosities (arrow). G , Insets from E , F have been digitally enlarged to show axonal varicosities (arrows). The bar graph in G shows the average number of varicosities per 100 µm of axon from N = 3 CON (45 mm total axon length) and three Lis1 KO (27 mm total axon length) mice. H , Kymographs were generated from time-lapse movies of LysoTracker labeled organelles in GFP-positive axons. The bar graph shows the percentage moving retrogradely in Lis1 KO and no flLis1 control cultures (CON). A total of 27 100 µm axon segments were analyzed from N = 2 CON and N = 2 Lis1 KO mice. A total of n = 521 control and n = 699 KO organelles were analyzed. I , Cultured DRG neurons prepared from intraperitoneally injected, no flLis1 control and Lis1 KO mice were immunostained with neuron-specific antibodies, and the percentage of neurons with growing axons was determined from N = 4 CON and N = 5 Lis1 KO mice. A total of n = 2219 control neurons and n = 2410 Lis1 KO neurons were analyzed. Bars in G-I indicate mean ± SD. Significance determined by Mann–Whitney test ( G ), Student’s t test ( H , I ), * p < 0.05, *** p < 0.001 (see for details). Scale bars: 20 µm ( A , D , E ), 5 µm ( B ), and 50 µm ( I ).

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques: Cell Culture, Control, Expressing, Injection, Clinical Proteomics, Generated, Labeling, MANN-WHITNEY

Brainstem neurons in Lis1 KO mice exhibit signs of chromatolysis. A , A coronal section through the hindbrain on day 4 after the 2 × 8 mg tamoxifen regimen shows extensive recombination in the ventral brainstem containing cardiorespiratory centers. White circles indicate the region used in the analyses of chromatolysis. B , C , Sections were stained with toluidine blue to determine the size and position of the nucleus in neurons in the indicated regions. The neurons in B are from a no flLis1 control mouse. The neurons in C are from a Lis1 KO animal. D , A nuclear enlargement index (see Materials and Methods) was used to compare nuclear enlargement in no flLis1 controls (CON) and Lis1 KO (KO). E , The histogram shows the distribution of this index in CON and Lis1 KO neurons. F , The position of the nucleus within the soma was also determined using the centroid displacement index (see Materials and Methods). This involves determining the centroid position of both the nucleus and soma and calculating the total displacement distance (µm) of the nuclear centroid from the somal centroid. G , Histogram showing the distribution of CDI found in CON and KO neurons. Bars indicate mean ± SD. Brainstem sections from three no flLis1 control and four Lis1 KO mice were used in the chromatolysis study. This includes analysis of 331 control neurons and 583 Lis1 KO neurons. Significance determined by Student’s t test ( D ), or Mann–Whitney test ( F ); *** p < 0.001 (see for details). Scale bars: 1 mm ( A ) and 10 µm ( B , C ).

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Brainstem neurons in Lis1 KO mice exhibit signs of chromatolysis. A , A coronal section through the hindbrain on day 4 after the 2 × 8 mg tamoxifen regimen shows extensive recombination in the ventral brainstem containing cardiorespiratory centers. White circles indicate the region used in the analyses of chromatolysis. B , C , Sections were stained with toluidine blue to determine the size and position of the nucleus in neurons in the indicated regions. The neurons in B are from a no flLis1 control mouse. The neurons in C are from a Lis1 KO animal. D , A nuclear enlargement index (see Materials and Methods) was used to compare nuclear enlargement in no flLis1 controls (CON) and Lis1 KO (KO). E , The histogram shows the distribution of this index in CON and Lis1 KO neurons. F , The position of the nucleus within the soma was also determined using the centroid displacement index (see Materials and Methods). This involves determining the centroid position of both the nucleus and soma and calculating the total displacement distance (µm) of the nuclear centroid from the somal centroid. G , Histogram showing the distribution of CDI found in CON and KO neurons. Bars indicate mean ± SD. Brainstem sections from three no flLis1 control and four Lis1 KO mice were used in the chromatolysis study. This includes analysis of 331 control neurons and 583 Lis1 KO neurons. Significance determined by Student’s t test ( D ), or Mann–Whitney test ( F ); *** p < 0.001 (see for details). Scale bars: 1 mm ( A ) and 10 µm ( B , C ).

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques: Staining, Control, MANN-WHITNEY

Comparing the effect of Lis1 KO in brainstem and heart. A , Sagittal brain sections of Lis1 KO mice 5 d after the initial injection of either five injections of 2 mg tamoxifen (top) or two injections of 8 mg tamoxifen (bottom). The 2 × 8 mg treatment resulted in much higher GFP expression than the 5 × 2 mg treatment, particularly in the brainstem and cerebellum. B , Lis1 mRNA levels normalized to B2M mRNA levels from brainstem of no Cre control mice injected with 2 × 8 mg tamoxifen (CON), and Lis1 KO mice injected with either 5 × 2 or 2 × 8 mg tamoxifen. Lis1 mRNA levels were significantly decreased in brainstem of 2 × 8 mg animals, but not 5 × 2 mg animals, relative to no Cre controls, 5 d after initial injection. C , Sections of heart from 5 × 2 mg (top)- and 2 × 8 mg (bottom)-treated Lis1 KO mice. Both the 2 × 8 and 5 × 2 mg treatments resulted in similar levels of GFP expression in heart. D , Lis1 mRNA levels normalized to B2M mRNA levels from heart of 2 × 8 mg-injected no Cre control (CON)-, 5 × 2 mg-, and 2 × 8 mg-treated mice. Lis1 mRNA levels were reduced significantly in both the 5 × 2 mg- and 2 × 8 mg-treated mice relative to the no Cre control but were not significantly different from each other. E , Western blotting of brainstem and heart lysates from cardiomyocyte-specific Myh6 KO mice show reduced levels of Lis1 protein in heart, but not brainstem compared to no Cre control mice (CON). Dynein intermediate chain (DIC) was used as a loading control. F , Whole mount brain (right) and heart (left) from Myh6 KO mouse show recombination (GFP) in heart but not brain. Data in A , C , E , F are representative images from N = 3 mice for each genotype. The RNA quantification in B , D represent mean of data from N = 3 animals of each treatment and genotype ± SD. Significance in B , D determined by one-way ANOVA; * p < 0.05, *** p < 0.001 (see for details). Scale bars: 5 mm ( A , C ) and 2 mm ( F ).

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Comparing the effect of Lis1 KO in brainstem and heart. A , Sagittal brain sections of Lis1 KO mice 5 d after the initial injection of either five injections of 2 mg tamoxifen (top) or two injections of 8 mg tamoxifen (bottom). The 2 × 8 mg treatment resulted in much higher GFP expression than the 5 × 2 mg treatment, particularly in the brainstem and cerebellum. B , Lis1 mRNA levels normalized to B2M mRNA levels from brainstem of no Cre control mice injected with 2 × 8 mg tamoxifen (CON), and Lis1 KO mice injected with either 5 × 2 or 2 × 8 mg tamoxifen. Lis1 mRNA levels were significantly decreased in brainstem of 2 × 8 mg animals, but not 5 × 2 mg animals, relative to no Cre controls, 5 d after initial injection. C , Sections of heart from 5 × 2 mg (top)- and 2 × 8 mg (bottom)-treated Lis1 KO mice. Both the 2 × 8 and 5 × 2 mg treatments resulted in similar levels of GFP expression in heart. D , Lis1 mRNA levels normalized to B2M mRNA levels from heart of 2 × 8 mg-injected no Cre control (CON)-, 5 × 2 mg-, and 2 × 8 mg-treated mice. Lis1 mRNA levels were reduced significantly in both the 5 × 2 mg- and 2 × 8 mg-treated mice relative to the no Cre control but were not significantly different from each other. E , Western blotting of brainstem and heart lysates from cardiomyocyte-specific Myh6 KO mice show reduced levels of Lis1 protein in heart, but not brainstem compared to no Cre control mice (CON). Dynein intermediate chain (DIC) was used as a loading control. F , Whole mount brain (right) and heart (left) from Myh6 KO mouse show recombination (GFP) in heart but not brain. Data in A , C , E , F are representative images from N = 3 mice for each genotype. The RNA quantification in B , D represent mean of data from N = 3 animals of each treatment and genotype ± SD. Significance in B , D determined by one-way ANOVA; * p < 0.05, *** p < 0.001 (see for details). Scale bars: 5 mm ( A , C ) and 2 mm ( F ).

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques: Injection, Expressing, Control, Western Blot

Mouse strains used in these studies

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Mouse strains used in these studies

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques: Control

Lis1 protein is expressed in adult mouse tissues. A total of 1 µg of tissue lysates was analyzed using the Wes Simple Western System. Brain extracts from E19 were loaded as a positive control. All other extracts are from two-month-old animals. The size-based separation is processed by Compass software and displayed as virtual blots/gels. A , Immune detection of Lis1 in protein samples, depicted in a virtual immunoblot generated by the system. B , Total protein detection, visualized by a virtual Coomassie gel generated by the system. These blots are representative of three experimental repeats ( N = 3).

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Lis1 protein is expressed in adult mouse tissues. A total of 1 µg of tissue lysates was analyzed using the Wes Simple Western System. Brain extracts from E19 were loaded as a positive control. All other extracts are from two-month-old animals. The size-based separation is processed by Compass software and displayed as virtual blots/gels. A , Immune detection of Lis1 in protein samples, depicted in a virtual immunoblot generated by the system. B , Total protein detection, visualized by a virtual Coomassie gel generated by the system. These blots are representative of three experimental repeats ( N = 3).

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques: Simple Western, Positive Control, Software, Western Blot, Generated

Cre-dependent recombination in the brain after tamoxifen injection. All data in this figure are representative of observations from a minimum of N = 4 animals of each genotype. A , On day 5 after the 2 × 8 mg tamoxifen regimen, no Cre control brains (CON, day 5) had bright dtTomato fluorescence (top left panel), but no GFP fluorescence indicative of recombination (lower left panel). Lis1 KO mice (Lis1 KO, day 5) showed reduced dtTomato fluorescence (top right panel) and expressed EGFP primarily in the hindbrain, indicating that Cre activity was pronounced in this brain region (lower right panel). B , Lis1 KO het mice [Lis1 KO (Het), day 21], which showed no sign of neurologic problems through day 21 after the injection had substantial GFP expression throughout the brain at that time. C , A sagittal section of a Lis1 KO brain on day 5 (Lis1 KO, day 5) shows mosaic recombination in midbrain (white arrow), hindbrain (magenta arrow), and cerebellum (blue arrow), with widely scattered GFP-positive cells in cortex and hippocampus. Recombination also occurs in olfactory bulb (asterisk). D , Using higher magnification, GFP-positive cells in the midbrain can be seen interspersed with cells that have not yet undergone recombination. E , Fibers labeled with GFP are clearly visible in the brainstem. F , Lis1 expression is reduced in extracts from brainstem and cerebellum of Lis1 KO mice compared to no Cre controls. Scale bars: 5 mm ( A–C ), 100 µm ( D ), and 20 µm ( E ).

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Cre-dependent recombination in the brain after tamoxifen injection. All data in this figure are representative of observations from a minimum of N = 4 animals of each genotype. A , On day 5 after the 2 × 8 mg tamoxifen regimen, no Cre control brains (CON, day 5) had bright dtTomato fluorescence (top left panel), but no GFP fluorescence indicative of recombination (lower left panel). Lis1 KO mice (Lis1 KO, day 5) showed reduced dtTomato fluorescence (top right panel) and expressed EGFP primarily in the hindbrain, indicating that Cre activity was pronounced in this brain region (lower right panel). B , Lis1 KO het mice [Lis1 KO (Het), day 21], which showed no sign of neurologic problems through day 21 after the injection had substantial GFP expression throughout the brain at that time. C , A sagittal section of a Lis1 KO brain on day 5 (Lis1 KO, day 5) shows mosaic recombination in midbrain (white arrow), hindbrain (magenta arrow), and cerebellum (blue arrow), with widely scattered GFP-positive cells in cortex and hippocampus. Recombination also occurs in olfactory bulb (asterisk). D , Using higher magnification, GFP-positive cells in the midbrain can be seen interspersed with cells that have not yet undergone recombination. E , Fibers labeled with GFP are clearly visible in the brainstem. F , Lis1 expression is reduced in extracts from brainstem and cerebellum of Lis1 KO mice compared to no Cre controls. Scale bars: 5 mm ( A–C ), 100 µm ( D ), and 20 µm ( E ).

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques: Injection, Control, Fluorescence, Activity Assay, Expressing, Labeling

Statistics used in the indicated experiments

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Statistics used in the indicated experiments

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques:

Lis1 KO impacts axonal function in adult mouse DRG neurons. A , Cultured DRG neurons from no Cre control exposed to 4-OH tamoxifen for 5 d expressed only tdTomato (red) showing no signs of recombination. B , In contrast, Lis1 KO neurons had strong GFP expression (green) demonstrating recombination. C , 4-OH tamoxifen reduced Lis1 protein levels in Lis1 KO neurons relative to no Cre control neurons (CON). D , Intraperitoneal injection of 2 × 8 mg tamoxifen in Lis1 KO mice resulted in GFP expression in intact DRGs after 4 d. Arrows point to DRG plasma membranes. E , Cultured DRG neurons prepared from intraperitoneally injected, no Cre control animals expressed only tdTomato (red). NF (blue) was prominent along axon shafts (white arrow) but less prominent in axon terminals (arrowhead). F , DRG neurons prepared from intraperitoneally injected Lis1 KO mice continued to express GFP (green) in culture, and NF (blue) was most prominent in distal axons and enriched in in varicosities (arrow). G , Insets from E , F have been digitally enlarged to show axonal varicosities (arrows). The bar graph in G shows the average number of varicosities per 100 µm of axon from N = 3 CON (45 mm total axon length) and three Lis1 KO (27 mm total axon length) mice. H , Kymographs were generated from time-lapse movies of LysoTracker labeled organelles in GFP-positive axons. The bar graph shows the percentage moving retrogradely in Lis1 KO and no flLis1 control cultures (CON). A total of 27 100 µm axon segments were analyzed from N = 2 CON and N = 2 Lis1 KO mice. A total of n = 521 control and n = 699 KO organelles were analyzed. I , Cultured DRG neurons prepared from intraperitoneally injected, no flLis1 control and Lis1 KO mice were immunostained with neuron-specific antibodies, and the percentage of neurons with growing axons was determined from N = 4 CON and N = 5 Lis1 KO mice. A total of n = 2219 control neurons and n = 2410 Lis1 KO neurons were analyzed. Bars in G-I indicate mean ± SD. Significance determined by Mann–Whitney test ( G ), Student’s t test ( H , I ), * p < 0.05, *** p < 0.001 (see for details). Scale bars: 20 µm ( A , D , E ), 5 µm ( B ), and 50 µm ( I ).

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Lis1 KO impacts axonal function in adult mouse DRG neurons. A , Cultured DRG neurons from no Cre control exposed to 4-OH tamoxifen for 5 d expressed only tdTomato (red) showing no signs of recombination. B , In contrast, Lis1 KO neurons had strong GFP expression (green) demonstrating recombination. C , 4-OH tamoxifen reduced Lis1 protein levels in Lis1 KO neurons relative to no Cre control neurons (CON). D , Intraperitoneal injection of 2 × 8 mg tamoxifen in Lis1 KO mice resulted in GFP expression in intact DRGs after 4 d. Arrows point to DRG plasma membranes. E , Cultured DRG neurons prepared from intraperitoneally injected, no Cre control animals expressed only tdTomato (red). NF (blue) was prominent along axon shafts (white arrow) but less prominent in axon terminals (arrowhead). F , DRG neurons prepared from intraperitoneally injected Lis1 KO mice continued to express GFP (green) in culture, and NF (blue) was most prominent in distal axons and enriched in in varicosities (arrow). G , Insets from E , F have been digitally enlarged to show axonal varicosities (arrows). The bar graph in G shows the average number of varicosities per 100 µm of axon from N = 3 CON (45 mm total axon length) and three Lis1 KO (27 mm total axon length) mice. H , Kymographs were generated from time-lapse movies of LysoTracker labeled organelles in GFP-positive axons. The bar graph shows the percentage moving retrogradely in Lis1 KO and no flLis1 control cultures (CON). A total of 27 100 µm axon segments were analyzed from N = 2 CON and N = 2 Lis1 KO mice. A total of n = 521 control and n = 699 KO organelles were analyzed. I , Cultured DRG neurons prepared from intraperitoneally injected, no flLis1 control and Lis1 KO mice were immunostained with neuron-specific antibodies, and the percentage of neurons with growing axons was determined from N = 4 CON and N = 5 Lis1 KO mice. A total of n = 2219 control neurons and n = 2410 Lis1 KO neurons were analyzed. Bars in G-I indicate mean ± SD. Significance determined by Mann–Whitney test ( G ), Student’s t test ( H , I ), * p < 0.05, *** p < 0.001 (see for details). Scale bars: 20 µm ( A , D , E ), 5 µm ( B ), and 50 µm ( I ).

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques: Cell Culture, Control, Expressing, Injection, Clinical Proteomics, Generated, Labeling, MANN-WHITNEY

Brainstem neurons in Lis1 KO mice exhibit signs of chromatolysis. A , A coronal section through the hindbrain on day 4 after the 2 × 8 mg tamoxifen regimen shows extensive recombination in the ventral brainstem containing cardiorespiratory centers. White circles indicate the region used in the analyses of chromatolysis. B , C , Sections were stained with toluidine blue to determine the size and position of the nucleus in neurons in the indicated regions. The neurons in B are from a no flLis1 control mouse. The neurons in C are from a Lis1 KO animal. D , A nuclear enlargement index (see Materials and Methods) was used to compare nuclear enlargement in no flLis1 controls (CON) and Lis1 KO (KO). E , The histogram shows the distribution of this index in CON and Lis1 KO neurons. F , The position of the nucleus within the soma was also determined using the centroid displacement index (see Materials and Methods). This involves determining the centroid position of both the nucleus and soma and calculating the total displacement distance (µm) of the nuclear centroid from the somal centroid. G , Histogram showing the distribution of CDI found in CON and KO neurons. Bars indicate mean ± SD. Brainstem sections from three no flLis1 control and four Lis1 KO mice were used in the chromatolysis study. This includes analysis of 331 control neurons and 583 Lis1 KO neurons. Significance determined by Student’s t test ( D ), or Mann–Whitney test ( F ); *** p < 0.001 (see for details). Scale bars: 1 mm ( A ) and 10 µm ( B , C ).

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Brainstem neurons in Lis1 KO mice exhibit signs of chromatolysis. A , A coronal section through the hindbrain on day 4 after the 2 × 8 mg tamoxifen regimen shows extensive recombination in the ventral brainstem containing cardiorespiratory centers. White circles indicate the region used in the analyses of chromatolysis. B , C , Sections were stained with toluidine blue to determine the size and position of the nucleus in neurons in the indicated regions. The neurons in B are from a no flLis1 control mouse. The neurons in C are from a Lis1 KO animal. D , A nuclear enlargement index (see Materials and Methods) was used to compare nuclear enlargement in no flLis1 controls (CON) and Lis1 KO (KO). E , The histogram shows the distribution of this index in CON and Lis1 KO neurons. F , The position of the nucleus within the soma was also determined using the centroid displacement index (see Materials and Methods). This involves determining the centroid position of both the nucleus and soma and calculating the total displacement distance (µm) of the nuclear centroid from the somal centroid. G , Histogram showing the distribution of CDI found in CON and KO neurons. Bars indicate mean ± SD. Brainstem sections from three no flLis1 control and four Lis1 KO mice were used in the chromatolysis study. This includes analysis of 331 control neurons and 583 Lis1 KO neurons. Significance determined by Student’s t test ( D ), or Mann–Whitney test ( F ); *** p < 0.001 (see for details). Scale bars: 1 mm ( A ) and 10 µm ( B , C ).

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques: Staining, Control, MANN-WHITNEY

Comparing the effect of Lis1 KO in brainstem and heart. A , Sagittal brain sections of Lis1 KO mice 5 d after the initial injection of either five injections of 2 mg tamoxifen (top) or two injections of 8 mg tamoxifen (bottom). The 2 × 8 mg treatment resulted in much higher GFP expression than the 5 × 2 mg treatment, particularly in the brainstem and cerebellum. B , Lis1 mRNA levels normalized to B2M mRNA levels from brainstem of no Cre control mice injected with 2 × 8 mg tamoxifen (CON), and Lis1 KO mice injected with either 5 × 2 or 2 × 8 mg tamoxifen. Lis1 mRNA levels were significantly decreased in brainstem of 2 × 8 mg animals, but not 5 × 2 mg animals, relative to no Cre controls, 5 d after initial injection. C , Sections of heart from 5 × 2 mg (top)- and 2 × 8 mg (bottom)-treated Lis1 KO mice. Both the 2 × 8 and 5 × 2 mg treatments resulted in similar levels of GFP expression in heart. D , Lis1 mRNA levels normalized to B2M mRNA levels from heart of 2 × 8 mg-injected no Cre control (CON)-, 5 × 2 mg-, and 2 × 8 mg-treated mice. Lis1 mRNA levels were reduced significantly in both the 5 × 2 mg- and 2 × 8 mg-treated mice relative to the no Cre control but were not significantly different from each other. E , Western blotting of brainstem and heart lysates from cardiomyocyte-specific Myh6 KO mice show reduced levels of Lis1 protein in heart, but not brainstem compared to no Cre control mice (CON). Dynein intermediate chain (DIC) was used as a loading control. F , Whole mount brain (right) and heart (left) from Myh6 KO mouse show recombination (GFP) in heart but not brain. Data in A , C , E , F are representative images from N = 3 mice for each genotype. The RNA quantification in B , D represent mean of data from N = 3 animals of each treatment and genotype ± SD. Significance in B , D determined by one-way ANOVA; * p < 0.05, *** p < 0.001 (see for details). Scale bars: 5 mm ( A , C ) and 2 mm ( F ).

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Comparing the effect of Lis1 KO in brainstem and heart. A , Sagittal brain sections of Lis1 KO mice 5 d after the initial injection of either five injections of 2 mg tamoxifen (top) or two injections of 8 mg tamoxifen (bottom). The 2 × 8 mg treatment resulted in much higher GFP expression than the 5 × 2 mg treatment, particularly in the brainstem and cerebellum. B , Lis1 mRNA levels normalized to B2M mRNA levels from brainstem of no Cre control mice injected with 2 × 8 mg tamoxifen (CON), and Lis1 KO mice injected with either 5 × 2 or 2 × 8 mg tamoxifen. Lis1 mRNA levels were significantly decreased in brainstem of 2 × 8 mg animals, but not 5 × 2 mg animals, relative to no Cre controls, 5 d after initial injection. C , Sections of heart from 5 × 2 mg (top)- and 2 × 8 mg (bottom)-treated Lis1 KO mice. Both the 2 × 8 and 5 × 2 mg treatments resulted in similar levels of GFP expression in heart. D , Lis1 mRNA levels normalized to B2M mRNA levels from heart of 2 × 8 mg-injected no Cre control (CON)-, 5 × 2 mg-, and 2 × 8 mg-treated mice. Lis1 mRNA levels were reduced significantly in both the 5 × 2 mg- and 2 × 8 mg-treated mice relative to the no Cre control but were not significantly different from each other. E , Western blotting of brainstem and heart lysates from cardiomyocyte-specific Myh6 KO mice show reduced levels of Lis1 protein in heart, but not brainstem compared to no Cre control mice (CON). Dynein intermediate chain (DIC) was used as a loading control. F , Whole mount brain (right) and heart (left) from Myh6 KO mouse show recombination (GFP) in heart but not brain. Data in A , C , E , F are representative images from N = 3 mice for each genotype. The RNA quantification in B , D represent mean of data from N = 3 animals of each treatment and genotype ± SD. Significance in B , D determined by one-way ANOVA; * p < 0.05, *** p < 0.001 (see for details). Scale bars: 5 mm ( A , C ) and 2 mm ( F ).

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques: Injection, Expressing, Control, Western Blot

Mouse strains used in these studies

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Mouse strains used in these studies

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques: Control

Lis1 protein is expressed in adult mouse tissues. A total of 1 µg of tissue lysates was analyzed using the Wes Simple Western System. Brain extracts from E19 were loaded as a positive control. All other extracts are from two-month-old animals. The size-based separation is processed by Compass software and displayed as virtual blots/gels. A , Immune detection of Lis1 in protein samples, depicted in a virtual immunoblot generated by the system. B , Total protein detection, visualized by a virtual Coomassie gel generated by the system. These blots are representative of three experimental repeats ( N = 3).

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Lis1 protein is expressed in adult mouse tissues. A total of 1 µg of tissue lysates was analyzed using the Wes Simple Western System. Brain extracts from E19 were loaded as a positive control. All other extracts are from two-month-old animals. The size-based separation is processed by Compass software and displayed as virtual blots/gels. A , Immune detection of Lis1 in protein samples, depicted in a virtual immunoblot generated by the system. B , Total protein detection, visualized by a virtual Coomassie gel generated by the system. These blots are representative of three experimental repeats ( N = 3).

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques: Simple Western, Positive Control, Software, Western Blot, Generated

Cre-dependent recombination in the brain after tamoxifen injection. All data in this figure are representative of observations from a minimum of N = 4 animals of each genotype. A , On day 5 after the 2 × 8 mg tamoxifen regimen, no Cre control brains (CON, day 5) had bright dtTomato fluorescence (top left panel), but no GFP fluorescence indicative of recombination (lower left panel). Lis1 KO mice (Lis1 KO, day 5) showed reduced dtTomato fluorescence (top right panel) and expressed EGFP primarily in the hindbrain, indicating that Cre activity was pronounced in this brain region (lower right panel). B , Lis1 KO het mice [Lis1 KO (Het), day 21], which showed no sign of neurologic problems through day 21 after the injection had substantial GFP expression throughout the brain at that time. C , A sagittal section of a Lis1 KO brain on day 5 (Lis1 KO, day 5) shows mosaic recombination in midbrain (white arrow), hindbrain (magenta arrow), and cerebellum (blue arrow), with widely scattered GFP-positive cells in cortex and hippocampus. Recombination also occurs in olfactory bulb (asterisk). D , Using higher magnification, GFP-positive cells in the midbrain can be seen interspersed with cells that have not yet undergone recombination. E , Fibers labeled with GFP are clearly visible in the brainstem. F , Lis1 expression is reduced in extracts from brainstem and cerebellum of Lis1 KO mice compared to no Cre controls. Scale bars: 5 mm ( A–C ), 100 µm ( D ), and 20 µm ( E ).

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Cre-dependent recombination in the brain after tamoxifen injection. All data in this figure are representative of observations from a minimum of N = 4 animals of each genotype. A , On day 5 after the 2 × 8 mg tamoxifen regimen, no Cre control brains (CON, day 5) had bright dtTomato fluorescence (top left panel), but no GFP fluorescence indicative of recombination (lower left panel). Lis1 KO mice (Lis1 KO, day 5) showed reduced dtTomato fluorescence (top right panel) and expressed EGFP primarily in the hindbrain, indicating that Cre activity was pronounced in this brain region (lower right panel). B , Lis1 KO het mice [Lis1 KO (Het), day 21], which showed no sign of neurologic problems through day 21 after the injection had substantial GFP expression throughout the brain at that time. C , A sagittal section of a Lis1 KO brain on day 5 (Lis1 KO, day 5) shows mosaic recombination in midbrain (white arrow), hindbrain (magenta arrow), and cerebellum (blue arrow), with widely scattered GFP-positive cells in cortex and hippocampus. Recombination also occurs in olfactory bulb (asterisk). D , Using higher magnification, GFP-positive cells in the midbrain can be seen interspersed with cells that have not yet undergone recombination. E , Fibers labeled with GFP are clearly visible in the brainstem. F , Lis1 expression is reduced in extracts from brainstem and cerebellum of Lis1 KO mice compared to no Cre controls. Scale bars: 5 mm ( A–C ), 100 µm ( D ), and 20 µm ( E ).

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques: Injection, Control, Fluorescence, Activity Assay, Expressing, Labeling

Statistics used in the indicated experiments

Journal: eNeuro

Article Title: An Essential Postdevelopmental Role for Lis1 in Mice

doi: 10.1523/ENEURO.0350-17.2018

Figure Lengend Snippet: Statistics used in the indicated experiments

Article Snippet: Primary antibodies used are as follows: Lis1 rabbit polyclonal 484/485 ( ; WB: 1:500); Lis1 rabbit polyclonal (Wes: 1:25, WB: 1:500; Santa Cruz Biotechnology sc-15319, RRID: AB_2159891 ); ERK1 rabbit polyclonal (Wes: 1:100; Abcam ab109282, RRID: AB_10862274 ); a mix of the pan-axonal neurofilament (NF) mouse monoclonal cocktail (IF: 1:500; BioLegend 837904, RRID: AB_2566782 ); NF light, medium, and heavy chain chicken polyclonals (IF: 1:500; Aves NFL, NFM, and NFH, RRIDs: AB_2313553 , AB_2313554 , AB_2313552 ); and the NF 200-kDa mouse monoclonal, clone RT97 (IF: 1:500; Millipore CBL212, RRID: AB_93408 ) were used to label NFs; GAP43 rabbit polyclonal (IF: 1:500; Novus Biologicals, NB300-143, RRID: AB_10001196 ); beta-III tubulin chicken polyclonal (IF: 1:500; Millipore AB9354, RRID: AB_570918 ); peripherin chicken polyclonal (IF: 1:500; Abcam ab39374, RRID: AB_777207 ); choline acetyltransferase goat polyclonal (IF: 1:100; Millipore AB144P, RRID: AB_11214092 ); MAP2 chicken polyclonal (IF: 1:100; Abcam ab5392, RRID: AB_2138153 ); myelin basic protein chicken polyclonal (IF: 1:500; Aves MBP, RRID: AB_2313550 ); α-tubulin mouse monoclonal (WB: 1:2000; Sigma-Aldrich T5168, RRID: AB_477579 ); and dynein intermediate chain mouse monoclonal (WB: 1:1000; Santa Cruz Biotechnology sc-13524, RRID: AB_668849 ).

Techniques:

NAGK-Lis1 interaction during the cell cycle. PLA was carried out on fixed HEK293T cells using primary antibodies against NAGK and Lis1, subsequent ICC was performed using anti-tubulin antibody and then DAPI staining was done. In interphase (A) and prophase cells (B), NAGK-Lis1 interactions were found on nuclear membranes (arrowheads). In metaphase (C), anaphase (D), telophase (E) and cytokinesis (F) stages, PLA signals were observed on chromosomes and/or on MTs (red arrowheads), and in other cytoplasmic areas (white arrowheads). Scale bar; 5 μm.

Journal: Molecules and Cells

Article Title: N -Acetyl- D -Glucosamine Kinase Interacts with Dynein-Lis1-NudE1 Complex and Regulates Cell Division

doi: 10.14348/molcells.2016.0119

Figure Lengend Snippet: NAGK-Lis1 interaction during the cell cycle. PLA was carried out on fixed HEK293T cells using primary antibodies against NAGK and Lis1, subsequent ICC was performed using anti-tubulin antibody and then DAPI staining was done. In interphase (A) and prophase cells (B), NAGK-Lis1 interactions were found on nuclear membranes (arrowheads). In metaphase (C), anaphase (D), telophase (E) and cytokinesis (F) stages, PLA signals were observed on chromosomes and/or on MTs (red arrowheads), and in other cytoplasmic areas (white arrowheads). Scale bar; 5 μm.

Article Snippet: The following antibodies were used at the indicated dilutions: chicken polyclonal NAGK (1:1000 for ICC; GW22347, Sigma, USA); mouse monoclonal NAGK (1:10 for PLA; Santa Cruz Biotechnology, USA); rabbit polyclonal NAGK (1:50 for PLA; GeneTex, USA); rabbit polyclonal DYNLRB1/LC7 (1:50 for ICC and 1:25 for PLA; Proteintech Group, USA); mouse polyclonal dynein IC1 (1:25 for PLA; Santa Cruz Biotechnology); rabbit polyclonal Lis1 (Santa Cruz); rabbit polyclonal NudE1 (1:50 for ICC, Proteintech Group); rabbit polyclonal CENP-B (1:500; Abcam, UK); mouse monoclonal alpha-tubulin (1:10; broth preparation, Developmental Studies Hybridoma Bank, University of Iowa, USA); rabbit polyclonal lamin B (1:1000; Young In Frontier Inc., Korea).

Techniques: Staining

Interaction between NAGK and DYNLRB1-Lis1-NudE1 complex on nuclear envelopes during prophase nuclear envelope invagination (PNEI). (A) NAGK-DYNLRB-1 PLA and subsequent anti-lamin ICC was conducted on GT1-7 cells, which were then stained with DAPI. PLA signals were observed on nuclear envelopes (arrowheads) during nuclear envelope invagination which is indicated by concentrated lamin staining (green arrow). (B) NAGK-DYNLRB1 PLA (red dots), Lis1 ICC (green) and DAPI staining (blue) was performed on fixed HEK293T cells. PLA signals at nuclear envelopes colocalized with Lis1 ICC puncta are shown (original image and inset ; arrowheads). (C) NAGK-DYNLRB1 PLA, NudE1 ICC and DAPI staining were performed and colocalizations of NAGK-dynein interactions (red) with NudE1 (green) are shown on nuclear envelopes during PNEI (original image, and inset , arrowheads). Scale bar; 5 μm.

Journal: Molecules and Cells

Article Title: N -Acetyl- D -Glucosamine Kinase Interacts with Dynein-Lis1-NudE1 Complex and Regulates Cell Division

doi: 10.14348/molcells.2016.0119

Figure Lengend Snippet: Interaction between NAGK and DYNLRB1-Lis1-NudE1 complex on nuclear envelopes during prophase nuclear envelope invagination (PNEI). (A) NAGK-DYNLRB-1 PLA and subsequent anti-lamin ICC was conducted on GT1-7 cells, which were then stained with DAPI. PLA signals were observed on nuclear envelopes (arrowheads) during nuclear envelope invagination which is indicated by concentrated lamin staining (green arrow). (B) NAGK-DYNLRB1 PLA (red dots), Lis1 ICC (green) and DAPI staining (blue) was performed on fixed HEK293T cells. PLA signals at nuclear envelopes colocalized with Lis1 ICC puncta are shown (original image and inset ; arrowheads). (C) NAGK-DYNLRB1 PLA, NudE1 ICC and DAPI staining were performed and colocalizations of NAGK-dynein interactions (red) with NudE1 (green) are shown on nuclear envelopes during PNEI (original image, and inset , arrowheads). Scale bar; 5 μm.

Article Snippet: The following antibodies were used at the indicated dilutions: chicken polyclonal NAGK (1:1000 for ICC; GW22347, Sigma, USA); mouse monoclonal NAGK (1:10 for PLA; Santa Cruz Biotechnology, USA); rabbit polyclonal NAGK (1:50 for PLA; GeneTex, USA); rabbit polyclonal DYNLRB1/LC7 (1:50 for ICC and 1:25 for PLA; Proteintech Group, USA); mouse polyclonal dynein IC1 (1:25 for PLA; Santa Cruz Biotechnology); rabbit polyclonal Lis1 (Santa Cruz); rabbit polyclonal NudE1 (1:50 for ICC, Proteintech Group); rabbit polyclonal CENP-B (1:500; Abcam, UK); mouse monoclonal alpha-tubulin (1:10; broth preparation, Developmental Studies Hybridoma Bank, University of Iowa, USA); rabbit polyclonal lamin B (1:1000; Young In Frontier Inc., Korea).

Techniques: Staining

Colocalization of NAGK with Lis1 and NudE1 on nuclear envelopes after microtubule disruption in prophase cells. HEK293T cells were treated with nocodazole, double labeled with indicated antibodies and then stained with DAPI. (A) Tubulin signals disrupted by nocodazole and concentrated around centrosome are shown by red arrowheads (Tub/DAPI). In addition, colocalization of NAGK signal clusters (green arrowheads, NAGK) with tubulin are indicated by yellow arrowheads (merge). (B) ICCs with anti-NAGK (green) and anti-Lis1 (red) primary antibodies were performed on nocodazole treated prophase HEK293T cells, which were then stained with DAPI (blue). DAPI staining revealed invagination of prophase nuclear envelopes. Colocalizations of NAGK signals with Lis1 signals on nuclear envelopes are shown by arrowheads (boxed area enlarged inset ) during nuclear invagination. (C) Nocodazole treated prophase HEK293T cells were double stained with anti-NAGK (green) and anti-NudE1 (red) antibodies in a similar method. Colocalization between NAGK and NudE1 signals are shown by arrowheads on a nuclear envelope (arrowheads, inset ). (D) ICC doublelabeling with anti-NAGK (green) and anti-Lis1 (red) primary antibodies of control (not treated with nocodazole) HEK293T cells showed dispersed cytoplasmic signals (arrows). Scale bar; 5 μm.

Journal: Molecules and Cells

Article Title: N -Acetyl- D -Glucosamine Kinase Interacts with Dynein-Lis1-NudE1 Complex and Regulates Cell Division

doi: 10.14348/molcells.2016.0119

Figure Lengend Snippet: Colocalization of NAGK with Lis1 and NudE1 on nuclear envelopes after microtubule disruption in prophase cells. HEK293T cells were treated with nocodazole, double labeled with indicated antibodies and then stained with DAPI. (A) Tubulin signals disrupted by nocodazole and concentrated around centrosome are shown by red arrowheads (Tub/DAPI). In addition, colocalization of NAGK signal clusters (green arrowheads, NAGK) with tubulin are indicated by yellow arrowheads (merge). (B) ICCs with anti-NAGK (green) and anti-Lis1 (red) primary antibodies were performed on nocodazole treated prophase HEK293T cells, which were then stained with DAPI (blue). DAPI staining revealed invagination of prophase nuclear envelopes. Colocalizations of NAGK signals with Lis1 signals on nuclear envelopes are shown by arrowheads (boxed area enlarged inset ) during nuclear invagination. (C) Nocodazole treated prophase HEK293T cells were double stained with anti-NAGK (green) and anti-NudE1 (red) antibodies in a similar method. Colocalization between NAGK and NudE1 signals are shown by arrowheads on a nuclear envelope (arrowheads, inset ). (D) ICC doublelabeling with anti-NAGK (green) and anti-Lis1 (red) primary antibodies of control (not treated with nocodazole) HEK293T cells showed dispersed cytoplasmic signals (arrows). Scale bar; 5 μm.

Article Snippet: The following antibodies were used at the indicated dilutions: chicken polyclonal NAGK (1:1000 for ICC; GW22347, Sigma, USA); mouse monoclonal NAGK (1:10 for PLA; Santa Cruz Biotechnology, USA); rabbit polyclonal NAGK (1:50 for PLA; GeneTex, USA); rabbit polyclonal DYNLRB1/LC7 (1:50 for ICC and 1:25 for PLA; Proteintech Group, USA); mouse polyclonal dynein IC1 (1:25 for PLA; Santa Cruz Biotechnology); rabbit polyclonal Lis1 (Santa Cruz); rabbit polyclonal NudE1 (1:50 for ICC, Proteintech Group); rabbit polyclonal CENP-B (1:500; Abcam, UK); mouse monoclonal alpha-tubulin (1:10; broth preparation, Developmental Studies Hybridoma Bank, University of Iowa, USA); rabbit polyclonal lamin B (1:1000; Young In Frontier Inc., Korea).

Techniques: Disruption, Labeling, Staining, Control

NAGK interacted with dynein-Lis1-NudE1 complex on chromosomes in metaphase. (A) (a) NAGK-DYNLRB1 PLA and subsequent tubulin ICC and DAPI staining were performed in HEK293T cells. NAGK-dynein interactions (PLA) on chromosomes are indicated by red arrowheads, and in other cytoplasmic areas by white arrowheads. (b) Statistics: Majority of NAGK-dynein PLA signals were on chromosomes (53.84%) in metaphase. (B) (a) NAGK-Lis1 PLA was performed on HEK293T cells and followed by tubulin ICC and DAPI staining. NAGK-Lis1 interactions (PLA) were localized to chromosomes (red arrowheads) and in other cytoplasmic areas (white arrowheads). (b) NAGK-Lis1 interactions in meta-phase were occurred on chromosomes (64.28%) at a higher frequency compared to other cytoplasmic areas. NAGK-DYNLRB1 PLA followed by Lis1 (C) or NudE1 (D) immunostaining, and DAPI staining revealed colocalizations between NAGK-dynein complex (original image, inset , red arrowheads) and Lis1 (C, original image, inset , green arrowheads), and NudE1 (D, original image, inset , green arrowheads) in metaphase. Merged images show colocalization on chromosomes (C, D, original image, inset , yellow arrowheads). Scale bar; 5 μm.

Journal: Molecules and Cells

Article Title: N -Acetyl- D -Glucosamine Kinase Interacts with Dynein-Lis1-NudE1 Complex and Regulates Cell Division

doi: 10.14348/molcells.2016.0119

Figure Lengend Snippet: NAGK interacted with dynein-Lis1-NudE1 complex on chromosomes in metaphase. (A) (a) NAGK-DYNLRB1 PLA and subsequent tubulin ICC and DAPI staining were performed in HEK293T cells. NAGK-dynein interactions (PLA) on chromosomes are indicated by red arrowheads, and in other cytoplasmic areas by white arrowheads. (b) Statistics: Majority of NAGK-dynein PLA signals were on chromosomes (53.84%) in metaphase. (B) (a) NAGK-Lis1 PLA was performed on HEK293T cells and followed by tubulin ICC and DAPI staining. NAGK-Lis1 interactions (PLA) were localized to chromosomes (red arrowheads) and in other cytoplasmic areas (white arrowheads). (b) NAGK-Lis1 interactions in meta-phase were occurred on chromosomes (64.28%) at a higher frequency compared to other cytoplasmic areas. NAGK-DYNLRB1 PLA followed by Lis1 (C) or NudE1 (D) immunostaining, and DAPI staining revealed colocalizations between NAGK-dynein complex (original image, inset , red arrowheads) and Lis1 (C, original image, inset , green arrowheads), and NudE1 (D, original image, inset , green arrowheads) in metaphase. Merged images show colocalization on chromosomes (C, D, original image, inset , yellow arrowheads). Scale bar; 5 μm.

Article Snippet: The following antibodies were used at the indicated dilutions: chicken polyclonal NAGK (1:1000 for ICC; GW22347, Sigma, USA); mouse monoclonal NAGK (1:10 for PLA; Santa Cruz Biotechnology, USA); rabbit polyclonal NAGK (1:50 for PLA; GeneTex, USA); rabbit polyclonal DYNLRB1/LC7 (1:50 for ICC and 1:25 for PLA; Proteintech Group, USA); mouse polyclonal dynein IC1 (1:25 for PLA; Santa Cruz Biotechnology); rabbit polyclonal Lis1 (Santa Cruz); rabbit polyclonal NudE1 (1:50 for ICC, Proteintech Group); rabbit polyclonal CENP-B (1:500; Abcam, UK); mouse monoclonal alpha-tubulin (1:10; broth preparation, Developmental Studies Hybridoma Bank, University of Iowa, USA); rabbit polyclonal lamin B (1:1000; Young In Frontier Inc., Korea).

Techniques: Staining, Immunostaining

Diagram representing the interaction between NAGK, dynein, and the dynein adaptor proteins Lis1 and NudE1 in prophase and metaphase. (A) Prophase. In cells at prophase, centrosomes duplicate and MTs connect to nuclear envelope proteins, like Nup 133, with the aid of dynein complex and adaptor proteins, such as, Lis1, NudE1, and CENP-F ( , , ). Chromosomes are in a condensing state and centrosome-directed force (two red dotted arrows) generated by dynein movement creates nuclear envelope invagination in areas close to centrosomes, and causes breakdown in the nuclear envelope distal to centrosomes. We propose by binding to dynein and nuclear envelope, NAGK works as an adaptor to anchor dynein complex to the nuclear envelope and regulates its movement along MTs. (B) Metaphase. Centrosomes have moved to opposite ends of the cell, chromosomes align in the equatorial plane, and MT spindle fibers connect to KTs with the aid of dynein and its adaptor proteins. Our findings indicate that NAGK acts as an adaptor protein in the recruitment of dynein to KTs.

Journal: Molecules and Cells

Article Title: N -Acetyl- D -Glucosamine Kinase Interacts with Dynein-Lis1-NudE1 Complex and Regulates Cell Division

doi: 10.14348/molcells.2016.0119

Figure Lengend Snippet: Diagram representing the interaction between NAGK, dynein, and the dynein adaptor proteins Lis1 and NudE1 in prophase and metaphase. (A) Prophase. In cells at prophase, centrosomes duplicate and MTs connect to nuclear envelope proteins, like Nup 133, with the aid of dynein complex and adaptor proteins, such as, Lis1, NudE1, and CENP-F ( , , ). Chromosomes are in a condensing state and centrosome-directed force (two red dotted arrows) generated by dynein movement creates nuclear envelope invagination in areas close to centrosomes, and causes breakdown in the nuclear envelope distal to centrosomes. We propose by binding to dynein and nuclear envelope, NAGK works as an adaptor to anchor dynein complex to the nuclear envelope and regulates its movement along MTs. (B) Metaphase. Centrosomes have moved to opposite ends of the cell, chromosomes align in the equatorial plane, and MT spindle fibers connect to KTs with the aid of dynein and its adaptor proteins. Our findings indicate that NAGK acts as an adaptor protein in the recruitment of dynein to KTs.

Article Snippet: The following antibodies were used at the indicated dilutions: chicken polyclonal NAGK (1:1000 for ICC; GW22347, Sigma, USA); mouse monoclonal NAGK (1:10 for PLA; Santa Cruz Biotechnology, USA); rabbit polyclonal NAGK (1:50 for PLA; GeneTex, USA); rabbit polyclonal DYNLRB1/LC7 (1:50 for ICC and 1:25 for PLA; Proteintech Group, USA); mouse polyclonal dynein IC1 (1:25 for PLA; Santa Cruz Biotechnology); rabbit polyclonal Lis1 (Santa Cruz); rabbit polyclonal NudE1 (1:50 for ICC, Proteintech Group); rabbit polyclonal CENP-B (1:500; Abcam, UK); mouse monoclonal alpha-tubulin (1:10; broth preparation, Developmental Studies Hybridoma Bank, University of Iowa, USA); rabbit polyclonal lamin B (1:1000; Young In Frontier Inc., Korea).

Techniques: Generated, Binding Assay