goat polyclonal anti-map1b sc-8970  (Santa Cruz Biotechnology)

Journal: Traffic (Copenhagen, Denmark)

Article Title: Nuclear LC3 associates with slowly diffusing complexes that survey the nucleolus

doi: 10.1111/tra.12372

Figure Lengend Snippet: List of proteins co-immunoprecipitated with Venus-LC3 from nuclear fraction of HeLa cells *

Article Snippet: For validation of the MudPIT results, Western blots were probed with either mouse monoclonal anti-SQSTM1 (BD, 610832) at 1:2000 dilution or goat polyclonal anti-MAP1B (Santa Cruz, sc-8970) at 1:100 dilution in combination with rabbit polyclonal anti-MAP1LC3B (Novus, NB100-2220) at 1:5000.

Techniques: Binding Assay, Expressing, Cell Differentiation, RNA Binding Assay, Migration, Methylation, DNA Synthesis

Journal: Traffic (Copenhagen, Denmark)

Article Title: Nuclear LC3 associates with slowly diffusing complexes that survey the nucleolus

doi: 10.1111/tra.12372

Figure Lengend Snippet: (A) Schematic of experiment workflow. HeLa cells were transiently transfected with Venus or Venus-LC3 prior to subcellular fractionation. Nuclear or cytoplasmic extracts containing Venus or Venus-LC3 were incubated with GBP-Sepharose resin for 2h at 4° C in order to immunoprecipitate Venus or Venus-LC3 protein complexes (IP). IP fractions were washed 4 times (WO) and then eluted from the beads (elution). The input, unbound, wash, and eluted fractions were then analyzed by immunoblotting (C,D). Protein complexes obtained in elutions were partially resolved by SDS-PAGE followed by protein identification via MudPIT. Further validation of two of the proteins identified by MudPIT (MAP1B and SQSTM1) was performed by immunoblotting of IP fractions with anti-MAP1B and anti-SQSTM1 antibodies (C,D). (B) For quality control of subcellular fractionation, nuclear (N) and cytoplasmic (C) extracts of Venus or Venus-LC3 transfected HeLa cells were resolved by SDS-PAGE and western blotted with anti-HDAC2 (positive nuclear control) and β-Tubulin (positive cytoplasmic control) antibodies. IP fractions from (C) Nuclear and (D) Cytoplasmic HeLa extracts were electrophoretically resolved by SDS PAGE and immunoblotted with (i) anti-MAP1B, (ii) anti-SQSTM1, or (iii) anti-GFP antibodies. M, molecular weight markers.

Article Snippet: For validation of the MudPIT results, Western blots were probed with either mouse monoclonal anti-SQSTM1 (BD, 610832) at 1:2000 dilution or goat polyclonal anti-MAP1B (Santa Cruz, sc-8970) at 1:100 dilution in combination with rabbit polyclonal anti-MAP1LC3B (Novus, NB100-2220) at 1:5000.

Techniques: Transfection, Fractionation, Incubation, Western Blot, SDS Page, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: Tau Protects Microtubules in the Axon from Severing by Katanin

doi: 10.1523/JNEUROSCI.5392-05.2006

Figure Lengend Snippet: Quantitative analyses of microtubule mass in fibroblasts induced to overexpress katanin together with various MAP constructs. A, The quantitative data graphically on the average fluorescence intensity of microtubule total mass. GFP (GFP alone as the control); katanin (P60-katanin alone); Taxol (control cells cultured in Taxol-containing medium); Taxol plus katanin (P60-katanin transfected cells cultured in Taxol-containing medium); MAP2c (MAP2c alone); MAP2c plus katanin (the cells expressing MAP2c and P60-katanin); MAP1b (MAP1b alone); MAP1b plus katanin (cells expressing MAP1b and P60-katanin); MAP4 (MAP4 alone); MAP4 plus katanin (cells expressing MAP4 and P60-katanin); tau3R (tau-3R alone); tau3R plus katanin (cells expressing tau3R and P60-katanin); tau4R (cells expressing tau4R alone); tau4R plus P60-katanin (cells expressing tau4R and P60-katanin); tauMTBD (tauMTBD alone); tauMTBD plus katanin (cells expressing tauMTBD and P60-katanin); tauMTNBD (tauMTNBD alone); tauMTNBD plus katanin (cells expressing tauMTNBD and P60-katanin); tauFFT/6d (tauFFT/6d alone); tauFFT/6d plus katanin (cells expressing tauFFT/6d and P60-katanin). B, Bar graph showing calculations on the percentage diminution in microtubule mass in cells with the various treatments (MAP expression, Taxol, GFP alone) as a result of expression of P60-katanin, from high to low. Error bars represent SE. MT, Microtubule; AFU, arbitrary fluorescence units.

Article Snippet: Antibodies Antibodies (Abs) included the following: rabbit anti-P60-katanin polyclonal Ab (1:1000 for immunostaining) ( Karabay et al., 2004 ); rabbit anti-green fluorescent protein (anti-GFP) polyclonal Ab (1:500 for immunostaining; Abcam, Cambridge, UK); chicken polyclonal anti-myc tag Ab (1:500 for immunostaining; Abcam); mouse monoclonal anti-flag tag Ab (1:500 for immunostaining; Sigma, St. Louis, MO); unconjugated mouse monoclonal anti-β-tubulin Ab (1:200 for immunostaining; Sigma); cyanine 3 (Cy3)-conjugated mouse monoclonal anti-β-tubulin Ab (1:300 for immunostaining; Sigma); mouse monoclonal anti-MAP2 Ab (1:200 for immunostaining and 1:1000 for Western blot; Chemicon, Temecula, CA); goat polyclonal anti-MAP1b Ab (1:200 for immunostaining; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-MAP1b Ab (1:500,000 for Western blot; provided by I. Fisher, Drexel University, Philadelphia, PA); rabbit polyclonal anti-MAP4 Ab (1:100 for immunostaining; also called LHB; provided by J. C. Bulinski, Columbia University, New York, NY); rabbit polyclonal anti-tau Ab1 (1:2000 for immunostaining in experiments of RFL6 cells; also called CR; provided by G. Lee, University of Iowa, Iowa City, IA); rabbit polyclonal anti-tau Ab2 (1:500 for immunostaining in experiments of rat neurons and 1:1000 for Western blot; also called DD8; provided by G. Lee); mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:500 for Western blot; Ambion, Austin, TX); and a series of appropriate fluorescent and HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA).

Techniques: Construct, Fluorescence, Control, Cell Culture, Transfection, Expressing

Journal: The Journal of Neuroscience

Article Title: Tau Protects Microtubules in the Axon from Severing by Katanin

doi: 10.1523/JNEUROSCI.5392-05.2006

Figure Lengend Snippet: MAP2c protects microtubules from being severed by P60-katanin, but MAP1b does not. A, C, E, G, GFP–P60-katanin in green and immunostain for either plasmid-expressed MAP2 or MAP1b in red. B, D, F, H, Immunostains for microtubules. A, B, Cells that are not overexpressing P60-katanin. As shown in A and B, MAP1b expression does not cause abnormal bundling of microtubules. As shown in C and D, a cell overexpressing MAP1b and P60-katanin shows only a scattering of very short microtubules and severely reduced microtubule levels. (A neighboring cell not expressing MAP1b or P60-katanin displays a normal microtubule array). As shown in E and F, MAP2c expression causes the formation of dense bundles of microtubules. As shown in G and H, the microtubules in MAP2c-expressing cells show no indication of severing by overexpression of P60-katanin, and the microtubule mass is not reduced. Scale bar: A–H, 30 μm.

Article Snippet: Antibodies Antibodies (Abs) included the following: rabbit anti-P60-katanin polyclonal Ab (1:1000 for immunostaining) ( Karabay et al., 2004 ); rabbit anti-green fluorescent protein (anti-GFP) polyclonal Ab (1:500 for immunostaining; Abcam, Cambridge, UK); chicken polyclonal anti-myc tag Ab (1:500 for immunostaining; Abcam); mouse monoclonal anti-flag tag Ab (1:500 for immunostaining; Sigma, St. Louis, MO); unconjugated mouse monoclonal anti-β-tubulin Ab (1:200 for immunostaining; Sigma); cyanine 3 (Cy3)-conjugated mouse monoclonal anti-β-tubulin Ab (1:300 for immunostaining; Sigma); mouse monoclonal anti-MAP2 Ab (1:200 for immunostaining and 1:1000 for Western blot; Chemicon, Temecula, CA); goat polyclonal anti-MAP1b Ab (1:200 for immunostaining; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-MAP1b Ab (1:500,000 for Western blot; provided by I. Fisher, Drexel University, Philadelphia, PA); rabbit polyclonal anti-MAP4 Ab (1:100 for immunostaining; also called LHB; provided by J. C. Bulinski, Columbia University, New York, NY); rabbit polyclonal anti-tau Ab1 (1:2000 for immunostaining in experiments of RFL6 cells; also called CR; provided by G. Lee, University of Iowa, Iowa City, IA); rabbit polyclonal anti-tau Ab2 (1:500 for immunostaining in experiments of rat neurons and 1:1000 for Western blot; also called DD8; provided by G. Lee); mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:500 for Western blot; Ambion, Austin, TX); and a series of appropriate fluorescent and HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA).

Techniques: Plasmid Preparation, Expressing, Over Expression

Journal: The Journal of Neuroscience

Article Title: Tau Protects Microtubules in the Axon from Severing by Katanin

doi: 10.1523/JNEUROSCI.5392-05.2006

Figure Lengend Snippet: Quantitative analysis of MAP levels after treatment with siRNAs. Control siRNA and siRNAs to MAP2, MAP1b, and tau were transfected into hippocampal neurons. Cultures were fixed 1, 3, and 5 d, respectively, after transfection and immunostained for each MAP to evaluate its level as a result of the siRNA. Immunofluorescence indicates depletion of MAP2, MAP1b, and tau in siRNA-treated neurons (b, d, f), compared with control siRNA-treated neurons (B, D, F), is shown. A, C, E, The quantification of total fluorescence intensity within the soma revealing the progressive loss of MAP2, MAP1b, and tau depletion by siRNA. By day 5, the loss of MAP2, MAP1b, and tau in siRNA treated neurons are 95, 99, and 99%, respectively. G, Western blot of whole-cell extracts probed with the MAP2, MAP1b, and tau Ab, confirming the protein-lowering effect. GAPDH was used as the internal control. Error bars represent SE.

Article Snippet: Antibodies Antibodies (Abs) included the following: rabbit anti-P60-katanin polyclonal Ab (1:1000 for immunostaining) ( Karabay et al., 2004 ); rabbit anti-green fluorescent protein (anti-GFP) polyclonal Ab (1:500 for immunostaining; Abcam, Cambridge, UK); chicken polyclonal anti-myc tag Ab (1:500 for immunostaining; Abcam); mouse monoclonal anti-flag tag Ab (1:500 for immunostaining; Sigma, St. Louis, MO); unconjugated mouse monoclonal anti-β-tubulin Ab (1:200 for immunostaining; Sigma); cyanine 3 (Cy3)-conjugated mouse monoclonal anti-β-tubulin Ab (1:300 for immunostaining; Sigma); mouse monoclonal anti-MAP2 Ab (1:200 for immunostaining and 1:1000 for Western blot; Chemicon, Temecula, CA); goat polyclonal anti-MAP1b Ab (1:200 for immunostaining; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-MAP1b Ab (1:500,000 for Western blot; provided by I. Fisher, Drexel University, Philadelphia, PA); rabbit polyclonal anti-MAP4 Ab (1:100 for immunostaining; also called LHB; provided by J. C. Bulinski, Columbia University, New York, NY); rabbit polyclonal anti-tau Ab1 (1:2000 for immunostaining in experiments of RFL6 cells; also called CR; provided by G. Lee, University of Iowa, Iowa City, IA); rabbit polyclonal anti-tau Ab2 (1:500 for immunostaining in experiments of rat neurons and 1:1000 for Western blot; also called DD8; provided by G. Lee); mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:500 for Western blot; Ambion, Austin, TX); and a series of appropriate fluorescent and HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA).

Techniques: Control, Transfection, Immunofluorescence, Fluorescence, Western Blot

Journal: The Journal of Neuroscience

Article Title: Tau Protects Microtubules in the Axon from Severing by Katanin

doi: 10.1523/JNEUROSCI.5392-05.2006

Figure Lengend Snippet: Effects of depleting MAP2, MAP1b, and tau on the development of hippocampal neurons in culture. Hippocampal neurons were divided into four groups, treated with control siRNA or siRNA to MAP2, MAP1b, or tau, respectively. Replating of neurons was done 2 d after siRNA treatment. Two days after replating, neurons were fixed and stained for microtubules. A, Pie graphs for the ratio of stage I, II, and III hippocampal neurons in each group. There is no significant difference between the control group and the groups depleted of MAP2 or tau. In contrast, depleting MAP1b delayed their development (p < 0.01). B, The quantitative analysis of the lengths of minor processes and axons of neurons in each group. Compared with the control, the axonal lengths of neurons treated with MAP2, MAP1b, and tau siRNA are shorter (p < 0.01). The axonal length of neurons treated with MAP1b siRNA is the shortest among these three. C, The quantitative analysis of the minor process length of neurons in each group. No significant difference was discovered between either two groups (p > 0.05). D, The quantitative analysis of microtubule mass in cell bodies in each group. Only the depletion of MAP1b resulted in a diminution of microtubule levels (by 21%). The microtubule levels in the other MAP siRNA-treated groups were not reduced. 1, 2, 3, 4 in B–D are control siRNA-treated, MAP2-siRNA treated, MAP1b siRNA-treated, and tau siRNA-treated group, respectively. Error bars represent SE. MT, Microtubule; AFU, arbitrary fluorescence units.

Article Snippet: Antibodies Antibodies (Abs) included the following: rabbit anti-P60-katanin polyclonal Ab (1:1000 for immunostaining) ( Karabay et al., 2004 ); rabbit anti-green fluorescent protein (anti-GFP) polyclonal Ab (1:500 for immunostaining; Abcam, Cambridge, UK); chicken polyclonal anti-myc tag Ab (1:500 for immunostaining; Abcam); mouse monoclonal anti-flag tag Ab (1:500 for immunostaining; Sigma, St. Louis, MO); unconjugated mouse monoclonal anti-β-tubulin Ab (1:200 for immunostaining; Sigma); cyanine 3 (Cy3)-conjugated mouse monoclonal anti-β-tubulin Ab (1:300 for immunostaining; Sigma); mouse monoclonal anti-MAP2 Ab (1:200 for immunostaining and 1:1000 for Western blot; Chemicon, Temecula, CA); goat polyclonal anti-MAP1b Ab (1:200 for immunostaining; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-MAP1b Ab (1:500,000 for Western blot; provided by I. Fisher, Drexel University, Philadelphia, PA); rabbit polyclonal anti-MAP4 Ab (1:100 for immunostaining; also called LHB; provided by J. C. Bulinski, Columbia University, New York, NY); rabbit polyclonal anti-tau Ab1 (1:2000 for immunostaining in experiments of RFL6 cells; also called CR; provided by G. Lee, University of Iowa, Iowa City, IA); rabbit polyclonal anti-tau Ab2 (1:500 for immunostaining in experiments of rat neurons and 1:1000 for Western blot; also called DD8; provided by G. Lee); mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:500 for Western blot; Ambion, Austin, TX); and a series of appropriate fluorescent and HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA).

Techniques: Control, Staining, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Tau Protects Microtubules in the Axon from Severing by Katanin

doi: 10.1523/JNEUROSCI.5392-05.2006

Figure Lengend Snippet: Depletion of tau from cultured hippocampal neurons increases sensitivity to katanin-induced microtubule severing, whereas depletion of MAP1b or MAP2 does not. A–F, Microtubule immunostains of stage III hippocampal neurons. A, A neuron transfected with control siRNA and GFP. B, A neuron transfected with control siRNA and P60-katanin. C, A neuron transfected with MAP1b siRNA and GFP. D, A neuron transfected with MAP1b siRNA and P60-katanin. E, A neuron transfected with tau siRNA and GFP (axon is directed upwards in the panel). F, A neuron transfected with tau siRNA and P60-katanin. Note that the axons show no diminution in fluorescence intensity as a result of overexpression of P60-katanin in either control siRNA or MAP1b siRNA-treated neurons. However, a dramatic diminution was detected in tau-depleted neurons overexpressing P60-katanin. a–f, Glow-scale pseudocolored images of the axons in A–F, with white indicating the highest level, purple indicating the lowest level, and shades of red, orange, and yellow indicating intermediate levels. G, The quantification of microtubule mass in cell bodies, in minor processes, and in axons of stage III neurons in each group. Overexpression of P60-katanin in either control group or the three MAP siRNA-treated groups leads to a 33–40% diminution in microtubule mass from cell bodies (p < 0.01). Overexpression of P60-katanin in either control group or the three MAP siRNA-treated groups leads to a 60–71% diminution in microtubule mass from minor processes (p < 0.0001). The microtubules in axons of tau depleted neurons were very sensitive to overexpression of P60-katanin, leading to a 63% diminution (p < 0.001). The microtubules in axons of the other three groups showed no diminution as a result of the overexpression of P60-katanin (p > 0.05). Error bars represent SE. AFU, Arbitrary fluorescence units; Kat, katanin.

Article Snippet: Antibodies Antibodies (Abs) included the following: rabbit anti-P60-katanin polyclonal Ab (1:1000 for immunostaining) ( Karabay et al., 2004 ); rabbit anti-green fluorescent protein (anti-GFP) polyclonal Ab (1:500 for immunostaining; Abcam, Cambridge, UK); chicken polyclonal anti-myc tag Ab (1:500 for immunostaining; Abcam); mouse monoclonal anti-flag tag Ab (1:500 for immunostaining; Sigma, St. Louis, MO); unconjugated mouse monoclonal anti-β-tubulin Ab (1:200 for immunostaining; Sigma); cyanine 3 (Cy3)-conjugated mouse monoclonal anti-β-tubulin Ab (1:300 for immunostaining; Sigma); mouse monoclonal anti-MAP2 Ab (1:200 for immunostaining and 1:1000 for Western blot; Chemicon, Temecula, CA); goat polyclonal anti-MAP1b Ab (1:200 for immunostaining; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-MAP1b Ab (1:500,000 for Western blot; provided by I. Fisher, Drexel University, Philadelphia, PA); rabbit polyclonal anti-MAP4 Ab (1:100 for immunostaining; also called LHB; provided by J. C. Bulinski, Columbia University, New York, NY); rabbit polyclonal anti-tau Ab1 (1:2000 for immunostaining in experiments of RFL6 cells; also called CR; provided by G. Lee, University of Iowa, Iowa City, IA); rabbit polyclonal anti-tau Ab2 (1:500 for immunostaining in experiments of rat neurons and 1:1000 for Western blot; also called DD8; provided by G. Lee); mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:500 for Western blot; Ambion, Austin, TX); and a series of appropriate fluorescent and HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA).

Techniques: Cell Culture, Transfection, Control, Fluorescence, Over Expression