Journal: Stem cell research

Article Title: Generation of a heterozygous knockout human embryonic stem cell line for the OCIAD1 locus using CRISPR/CAS9 mediated targeting: BJNhem20-OCIAD1-CRISPR-39.

doi: 10.1016/j.scr.2015.12.037

Figure Lengend Snippet: Fig. 1. (A) Strategy for targeting OCIAD1 exon3 by CRISPR-CAS9. (B) Genomic targeting of OCIAD1 exon3 of BJNhem20-OCIAD1-CRISPR-39 identified by T7 endonuclease mediated digestion of high fidelity exon 3 amplicon detecting deletions in this clone. (C) Chromatograms showing sequence analysis to confirm heterozygous mutation in the targeted region of OCIAD1. (D) Western blot analysis showing decreased OCIAD1 levels in BJNhem20-OCIAD1-CRISPR-39. Graph represents standard error mean after analysis of three biological replicates using single factor ANOVA (P value 0.001). (E) Karyotype analysis of BJNhem20-OCIAD1-CRISPR-39. (F) Analysis of transcript levels of pluripotency marker genes Oct4, Nanog, TDGF and Sox2 by reverse transcription and polymerase chain reaction (RT-PCR) amplification. GAPDH was used to normalize transcript levels. (G) Immunostaining analysis of BJNhem20-OCIAD1-CRISPR-39 for depletion of OCIAD1 and for pluripotency markers Oct4, SSEA4 and TRA1-81 as indicated, compared to parental hESC line BJNhem20. (H) Differentiation analysis of BJNhem20-OCIAD1-CRIPSR-39: embryoid bodies stained to show differentiation to all the three germ layers by immunostaining for AFP, Brachyury and β-III tubulin marking the endoderm, mesoderm and ectoderm respectively.

Article Snippet: Primary antibodies used were against OCIAD1 (Abcam Ab91574), Oct4 (BD Biosciences BD611203), TRA1-81 and SSEA4 (kind gift from Peter Andrews, University of Sheffield, UK), Brachyury (Santa Cruz Biotech Cat no. SC-17743), β-III tubulin (Santacruz SC-51670), AFP (Sigma Chemical Pvt.

Techniques: CRISPR, Amplification, Sequencing, Mutagenesis, Western Blot, Marker, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Staining

Journal: bioRxiv

Article Title: Drosophila beta-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

doi: 10.1101/185827

Figure Lengend Snippet: The effect of growth temperature on transcript and protein levels of Drosophila tubulin genes in S2R+ cells was analyzed with microarrays (A), qRT-PCR (B,C) or immunoblotting (D,E). (A) Temperature dependence of signal intensities on microarray probes. (B) Transcript levels of significantly expressed tubulin genes at 25°C. ΔCT values are indicated. A ΔCT value of zero indicates a transcript level equal to average expression of the genes used for normalization; higher ΔCT values indicate a relative decrease in expression. (C) Temperature dependence of transcript levels of significantly expressed tubulin genes. For each gene, the level at 25°C was set to 1. (D) Total extracts of S2R+ cells grown at 11, 14, 25 or 30°C were analyzed after western blotting by Ponceau S and probing with the indicated antibodies. RL2 detects O-GlcNAc modification on proteins which is tightly correlated with growth temperature . Equal amounts of protein were loaded except for the first four lanes covering a dilution series of the 14°C extract as internal reference for quantification. (E) Signals obtained with anti-betaTubulin97EF and anti-alpha-tubulin from immunoblots as shown in (D) were quantified. Ponceau S staining intensity was used for normalization. Signal intensities observed at 25°C were set to 1. Bars indicate average (+/- s.d.; n = 3).

Article Snippet: Rabbit antibodies against beta-Tubulin 60D ( ) were kindly provided by R. Renkawitz-Pohl (University of Marburg, Marburg, Germany).

Techniques: Quantitative RT-PCR, Western Blot, Microarray, Expressing, Modification, Staining

Journal: bioRxiv

Article Title: Drosophila beta-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

doi: 10.1101/185827

Figure Lengend Snippet: Highlighted in yellow is the sequence of the peptide used for the production of a beta-Tubulin 97EF-specific antibody. The region encoded by the mutually exclusive exons 4B and 4C of beta-Tubulin 97EF are indicated by grey shading. The N- and C-terminal sequences (green shading) are less conserved than the core domains. These terminal sequences were present in the GST fusions which were expressed in bacteria for the characterization of various antibodies (see ).

Article Snippet: Rabbit antibodies against beta-Tubulin 60D ( ) were kindly provided by R. Renkawitz-Pohl (University of Marburg, Marburg, Germany).

Techniques: Sequencing, Bacteria

Journal: bioRxiv

Article Title: Drosophila beta-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

doi: 10.1101/185827

Figure Lengend Snippet: (A) Bacterial extracts containing GST fusion proteins with distinct N- and C-terminal extensions corresponding to the N and C-terminal tails of the indicated Drosophila beta-tubulin paralogs were resolved by SDS-PAGE and analyzed by Coomassie Blue staining. In addition, a control extract (-) from bacteria without an expression construct, as well as a dilution series with known amounts of molecular weight marker proteins was analyzed. (B) Extracts with GST beta-tubulin tail fusions (as in A) were analyzed by immunoblotting with the indicated antibodies. 100 ng of GST fusion protein per lane was loaded in case of the immunoblots with anti-GST, E7, E-10, and dN-17, while 5 ng was loaded for the analyses with anti-97EF and anti-60D. (C) Total extracts of S2R+ cells grown at 25°C were analyzed by immunoblotting with anti-beta-Tubulin 97EF and anti-beta-Tubulin E7. 20 μg of total cellular protein was loaded in case of the anti-97EF immunoblot and 10 μg in case of the E7 immunoblot. For determination of the amount of beta-Tubulin 97EF and of total beta-tubulin in S2R+ cells a dilution series of the bacterial extract containing a known amount of the GST fusion protein with beta-tubulin 97EF tails was resolved and analyzed on the same immunoblots.

Article Snippet: Rabbit antibodies against beta-Tubulin 60D ( ) were kindly provided by R. Renkawitz-Pohl (University of Marburg, Marburg, Germany).

Techniques: SDS Page, Staining, Control, Bacteria, Expressing, Construct, Molecular Weight, Marker, Western Blot

Journal: bioRxiv

Article Title: Drosophila beta-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

doi: 10.1101/185827

Figure Lengend Snippet: (A) Total extracts from w 1 embryos at different ages AED. Equal numbers of embryos at each age interval were loaded apart from a dilution series. An extract from betaTub97EF null mutants ( MiMIC ) was included. Ponceau S staining was used as a control for equal loading. Replicate immunoblots were probed with the indicated antibodies. (B) w 1 embryos were stained with anti-beta-Tubulin 97EF and a DNA stain (not shown). In addition, an embryo homozygous for Df(3R)BSC460 which deletes betaTub97EF is shown in the bottom panel ( Def 97EF ). Numbers on the left side indicate the displayed stages. Arrowheads indicate foregut (fg), midgut (mg), hindgut (hg) and posterior spiracles (ps). (C) Hindgut region (13-15 hours AED) labeled as indicated. Boundary cells (bc) are pointed out by arrowheads. (D) Larval hemocyte after double labeling with anti-beta-Tubulin 97EF and anti-alpha-tubulin. Scale bar 50 μm (B), 10 μm (C) and 5 μm (D).

Article Snippet: Rabbit antibodies against beta-Tubulin 60D ( ) were kindly provided by R. Renkawitz-Pohl (University of Marburg, Marburg, Germany).

Techniques: Staining, Control, Western Blot, Labeling

Journal: bioRxiv

Article Title: Drosophila beta-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

doi: 10.1101/185827

Figure Lengend Snippet: (A) Total extracts prepared from either late embryos (embryo), larvae or adults were resolved by SDS-PAGE and analyzed after Western blotting by Ponceau S staining and probing with the indicated antibodies. Samples were from either the w 1 control strain or betaTub97EF MiMIC null mutants (M for MiMIC ). Embryonic and larval extracts were prepared from samples aged for the indicated time (hours after AED). 36, 60, 84 and 108 hours AED correspond to mid-first, mid-second, early and late third instar, respectively. In case of adults, flies (0-1 day after eclosion) were separated according to sex before extract preparation. Equal amounts of protein were loaded in all lanes except for the last three where a dilution series of the w 1 larval extract (108 hours AED) was loaded as internal reference for quantification. (B,C) Immunostaining of the gut from w 1 and betaTub97EF MiMIC animals during third larval instar wandering stage (B) or from adults (C) with anti-beta-Tubulin 97EF and a DNA stain. Scale bar 20 μm.

Article Snippet: Rabbit antibodies against beta-Tubulin 60D ( ) were kindly provided by R. Renkawitz-Pohl (University of Marburg, Marburg, Germany).

Techniques: SDS Page, Western Blot, Staining, Control, Immunostaining

Journal: bioRxiv

Article Title: Drosophila beta-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

doi: 10.1101/185827

Figure Lengend Snippet: (A) Scheme of the betaTub97EF region. Transposon insertions ( 1877 , MiMIC and 3812 ) are indicated by triangles and exons by boxes with black filling in coding region, except for the two mutually exclusive exons 4B and 4C which are shown in grey. Small arrows below exons indicate the three primer pairs used for qRT-PCR. (B) betaTub97EF transcript levels were determined by qRT-PCR with embryos of the indicated genotypes ( Df = Df(3R)BSC460 ). Results with the three primer pairs were highly concordant in a given genotype and were therefore averaged. Transcript levels are indicated by bars (average +/- s.d., n = 3 primer pairs). Those of w 1 control embryos were set to 1. (C) Total extracts from embryos (15-16 hours AED) analyzed by immunoblotting with genotypes and antibodies indicated. Anti-alpha-tubulin and anti-PSTAIR were used as a mixture to probe the replicate immunoblot shown in the lower panel. (D) Hindgut region in embryos (14-15 hours AED) after immunostaining with genotypes and antibodies as indicated. Crumbs is maximally expressed in the boundary cells of the hindgut. Scale bar 20 μm.

Article Snippet: Rabbit antibodies against beta-Tubulin 60D ( ) were kindly provided by R. Renkawitz-Pohl (University of Marburg, Marburg, Germany).

Techniques: Quantitative RT-PCR, Control, Western Blot, Immunostaining

Journal: bioRxiv

Article Title: Drosophila beta-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

doi: 10.1101/185827

Figure Lengend Snippet: (A) qRT-PCR revealed that the major betaTub97EF transcript isoform with exon 4B is upregulated at low temperature, while the minor alternative transcript with exon 4C is downregulated. Expression levels at 25°C were set to 1. Bars indicate average (+/- s.d.; n = 3 technical replicates). (B) Immunoblotting revealed that beta-Tubulin 97EF protein is upregulated in embryos at low temperature, in contrast to beta-Tubulin 60D and alpha-tubulin. Bar diagram represents average immunoblot signal intensities at 14° and 30°C (+/- s.d.; n = 3 biological replicates). Ponceau S staining intensity was used for normalization. Intensities observed at 30°C were set to 1. (C-E) Upregulation of beta-Tubulin 97EF at low temperature is tissue-specific. (C) Experimental strategy for accurate quantification of anti-beta-Tubulin 97EF immunofluorescence signals. See text for further explanations. Scale bar 20 μm. (D) Confocal sections through hindgut illustrate labeling with the indicated antibodies and DNA staining in embryos of the indicated genotypes after development at 14°C and 30°C, respectively. Scale bar 20 μm. (E) Quantification of signal intensities obtained with anti-beta-Tubulin 97EF and anti-alpha-tubulin in the hindgut epithelium. Expression levels observed at 14°C were set to 1. Bars indicate average (+/- s.d., n = 6 embryos). (F-H) Temperature sensitivity of betaTub97EF mutant development. (F) Experimental strategy for the analyses at different temperatures. Egg collections were divided into aliquots and incubated at the indicated temperatures. One part (fix) was used for the determination of the number of unfertilized and overaged embryos. (G) Rate of larval hatching at different temperatures. (H) Rate of development to the adult stage at different temperatures. Bars indicate average (+/- s.d., n = 3). * p < 0.05, ** p< 0.01, *** p < 0.001 ( t test).

Article Snippet: Rabbit antibodies against beta-Tubulin 60D ( ) were kindly provided by R. Renkawitz-Pohl (University of Marburg, Marburg, Germany).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Staining, Immunofluorescence, Labeling, Mutagenesis, Incubation

Journal: bioRxiv

Article Title: Drosophila beta-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

doi: 10.1101/185827

Figure Lengend Snippet: Functional specialization was addressed by complementation experiments involving expression of beta-tubulin paralogs in betaTub56D mutants (A) as well as after ectopic expression (B,C). (A) The P-element insertions NP0949 and EY12330 within the betaTub56D region are indicated (triangles), as well as the intragenic breakpoint of the deficiency Df(2R)BSC782 . Exons are represented by boxes with black filling indicating coding region. Grey bars indicate the extent of development of betaTub56D NP0949 / Df(2R)BSC782 mutants with alphaTub84BP-GAL4 driven expression of the indicated UASt-beta-tubulin transgenes. Larval instar stage 1-3 (L1-L3), pupal stage (P) and adult stage (A). (B) Eye phenotype in adults after ey-GAL4 driven expression of the indicated UASt-beta-tubulin transgenes. (C,D) mat-4-GAL4VP16 was used for expression of the indicated UASp-beta-tubulin transgenes during oogenesis. (C) DNA staining in stage 10B egg chambers. Arrowheads point to some of the abnormal nurse cell nuclei. (D) Morphology of deposited eggs. Scale bar 50 μm.

Article Snippet: Rabbit antibodies against beta-Tubulin 60D ( ) were kindly provided by R. Renkawitz-Pohl (University of Marburg, Marburg, Germany).

Techniques: Functional Assay, Expressing, Staining

Journal: bioRxiv

Article Title: Drosophila beta-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

doi: 10.1101/185827

Figure Lengend Snippet: (A-D) Hemocytes were isolated from larvae with the indicated genotypes (97EF null = betaTub97EF MiMIC ; 56D null = betaTub56D NP0949 / Df(2R)BSC782 ; R-97EF and S-97EF = 56D null and Df(2R)BSC782/ balancer, respectively, with alphaTub84BP>betaTub97EF ; R-56D and S-56D = 56D null and Df(2R)BSC782/ balancer, respectively, with alphaTub84BP>betaTub56D ). (A) Hemocytes were double labeled with anti-beta-Tubulin 97E and anti-alpha-tubulin (left and middle columns) or only with anti-beta-tubulin (E7) (right column). Bar diagram represents average signal intensity (+/- s.d., n = 11). Intensities obtained in w 1 were set to 1. (B) Hemocytes from larvae with the indicated genotypes after development at the indicated temperatures were released onto the same cover slip to enforce identical fixation and staining conditions. Nuclear His2Av-mRFP fluorescence (insets) was used for hemocyte classification. Bar diagrams indicate average background corrected anti-beta-Tubulin 97EF signal intensities (+/- s.d., n = 20). Signals observed at 14°C in w 1 were set to 1. *** p < = 0.001 ( t test). (C) Representative still frames after time lapse imaging of hemocytes expressing EB1-GFP. Bar diagram indicates average speed of EB1-GFP comet movements (+/- s.d., n = 11). (D) Hemocytes of indicated genotypes were treated with colcemid for the indicated time before fixation and staining with anti-alpha-tubulin. Graph represents average signal intensity (+/- s.d., n = 6). Signals observed at time 0 hours were set to 1 for each genotype. Scale bars 5 μm.

Article Snippet: Rabbit antibodies against beta-Tubulin 60D ( ) were kindly provided by R. Renkawitz-Pohl (University of Marburg, Marburg, Germany).

Techniques: Isolation, Labeling, Staining, Fluorescence, Imaging, Expressing

Journal: bioRxiv

Article Title: Drosophila beta-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

doi: 10.1101/185827

Figure Lengend Snippet: (A-C) Embryos (13-14 hours AED) with or without betaTub97EF gene function ( g-tdTomato-CenpC and MiMIC , respectively) were exposed to vinblastine (+) or solvent only (-) before immunostaining with anti-alpha-tubulin. Sections illustrating the effect on anti-alpha-tubulin signal intensities in the hindgut and surrounding tissues (A) or in somatic muscles (B) are displayed. Scale bar 15 μm. (C) Bar diagram representing average anti-alpha-tubulin signal intensities in the hindgut and in the surrounding tissues (+/- s.d., n = 9). ** p < 0.01 ( t test). Signals in controls were set to 1.

Article Snippet: Rabbit antibodies against beta-Tubulin 60D ( ) were kindly provided by R. Renkawitz-Pohl (University of Marburg, Marburg, Germany).

Techniques: Solvent, Immunostaining, Muscles