Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: ( A ) Gene structure of the Rng105 genome, targeting vector, floxed Rng105 , and after excision of the floxed sequence. Arrowheads indicate PCR primers for genotyping. ( B ) PCR analysis of the indicated genotypes. ( C ) Male littermates at postnatal 9 ( P9 ) weeks. ( D ) Relative body weight of Camk2a-Cre;Rng105 f/f mice compared with Rng105 f/f mice. n = 5 and 3 (P0), 10 and 6 (P8), 27 and 22 (P30), 6 and 6 (P60), 9 and 8 (P90) for Rng105 f/f and Camk2a-Cre;Rng105 f/f mice, respectively. *p<0.05, **p<0.01, ****p<0.001 using two-way ANOVA followed by Student's t -test. Data are represented as the mean ± s.e.m. ( E ) Survival curves of Rng105 f/f and Camk2a-Cre;Rng105 f/f mice. The number of Camk2a-Cre;Rng105 f/f mice was 70.4% of Rng105 f/f mice at P30 (n = 402 and n = 283 for Rng105 f/f and Camk2a-Cre;Rng105 f/f , respectively). Thereafter, survival was analyzed in 13 Rng105 f/f mice and 26 Camk2a-Cre;Rng105 f/f mice. ( F ) Western blotting of indicated brain extracts from Rng105 f/f and Camk2a-Cre;Rng105 f/f mice for RNG105 and α-tubulin as a control. Bottom, quantification of the RNG105 band intensity normalized to α-tubulin. Data are represented as the mean ± s.e.m. with dot plots of individual values. n = 3. *p<0.05, ****p<0.001 using Student's t-test. ( G ) Staining of hippocampal slices with anti-RNG105 antibody and DAPI. SP, stratum pyramidale; SR, stratum radiatum. Scale bar, 50 µm. See also .

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques: Plasmid Preparation, Sequencing, Western Blot, Staining

Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: ( A ) Reduction of Rng105 transcripts in Camk2a-Cre;Rng105 f/f mice compared to Rng105 f/f mice. RNA-seq read coverage across the Rng105 genome for triplicate RNA samples from the hippocampus of Rng105 f/f and Camk2a-Cre;Rng105 f/f mice is shown. The perpendicular axis shows the number of reads standardized among the samples. Numbers indicate exon numbers of the Rng105 gene. Note that not only transcripts of exons 5–6 but also those of the other exons were reduced in amount in Camk2a-Cre;Rng105 f/f mice. ( B ) No truncated form of RNG105 protein is detected in the cerebrum of Camk2a-Cre;Rng105 f/f mice. Left, western blotting for truncated RNG105 (a.a. 1–122)-GFP (an arrowhead) expressed in CHO cells with an anti-GFP antibody. Right, western blotting of CHO cells expressing the truncated RNG105-GFP and the cerebral cortex from Rng105 f/f and Camk2a-Cre;Rng105 f/f mice with the anti-RNG105 antibody. The anti-RNG105 antibody detected the truncated RNG105-GFP (an arrowhead). Although the molecular mass of the truncated RNG105 (a.a. 1–122) is expected to be ~13 kDa, no such band was detected in the cerebrum of Camk2a-Cre;Rng105 f/f mice even after intensive exposure of the membrane to the detecting solution. Because of the intensive exposure, RNG105 band intensity was saturated, and bands other than RNG105 were also detected. An arrow denotes endogenous RNG105.

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques: RNA Sequencing Assay, Western Blot, Expressing

Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: ( A ) Fluorescence images of cultured hippocampal neurons from Rng105 f/f mice transfected with mCherry (left) or both mCherry and CMV-Cre (right), and immunostained for RNG105. Arrows indicate transfected neurons. Scale bar, 10 µm. ( B ) Immunofluorescence intensity of RNG105 in the transfected neurons normalized to that in neighboring mCherry-negative neurons. n = 24. Student's t-test, ****p=1.33E-6. ( C and D ) Density ( C ) and size ( D ) of dendritic spines in the transfected neurons. n = 15 neurons. Student's t-test, p=0.483 ( C ), *p=0.0407 ( D ). ( E ) Representative time-lapse images of dendritic spines before and after the induction of structural plasticity by two-photon glutamate uncaging. Asterisks indicate the location of uncaging. Scale bar, 1 µm. ( F ) Time course of spine volume change in stimulated spines. CHX, addition of cycloheximide; w/o Glu and 2P, mock experiments without caged glutamate or two-photon laser irradiation. n = 17, 7, 16, 6, 5 and 6, from top to bottom, respectively. ****p<0.001 using two-way repeated measures ANOVA. Data are represented as the mean ± s.e.m. ( G ) Average spine volume over 30–60 min in ( F ). *p<0.05, **p<0.01, ****p<0.001 using one-way ANOVA followed by Tukey-Kramer test. In B−D and G, data are represented by dot plots with the mean.

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques: Fluorescence, Cell Culture, Transfection, Immunofluorescence, Irradiation

Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: ( A ) Representative fEPSP traces evoked by different stimulation intensities. ( B ) Input-output (I/O) relationship between CA3 stimulation magnitude and CA1 fEPSP responses. n = 10 ( Rng105 f/f ) and 13 ( Camk2a-Cre;Rng105 f/f ). *p<0.05 using two-way repeated measures ANOVA followed by Bonferroni post-hoc t-test. ( C ) Representative paired-pulse ratio (PPR) with different inter-pulse intervals at CA3-CA1 synapses before (black) and after (red) LTP induction. The inset shows a magnified image. ( D ) Relationship between inter-pulse intervals and PPR. n = 7 ( Rng105 f/f ), 10 ( Camk2a-Cre;Rng105 f/f ), 7 ( Rng105 f/f , LTP), and 8 ( Camk2a-Cre;Rng105 f/f , LTP). *p<0.05, **p<0.01, between Camk2a-Cre;Rng105 f/f and Camk2a-Cre;Rng105 f/f (LTP), using two-way repeated measures ANOVA, Bartlett’s test, and one-way ANOVA followed by Tukey-Kramer test. There was no significant difference between the genotypes. ( E ) Representative fEPSP traces before (black) and after (red) LTP induction. ( F ) Time course of LTP in CA1 neurons. n = 7. Because the I/O response in Camk2a-Cre;Rng105 f/f mice was reduced to 45.1% of that in Rng105 f/f mice (at max value), the baseline fEPSP slope for Camk2a-Cre;Rng105 f/f mice is set at 45.1% of that for Rng105 f/f mice. Left and right scales are for Rng105 f/f mice and Camk2a-Cre;Rng105 f/f mice, respectively (the right scale is 45.1% of the left scale). ****p=0.0004 using Student's t-test at 140 min with the I/O normalization. Data in B, D and F are represented as the mean ± s.e.m.

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques:

Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: ( A ) Open field test. The number of line crossings during a 5 min trial on 3 consecutive days. n = 14 ( Rng105 f/f ), 9 ( Camk2a-Cre;Rng105 f/f ) and 10 ( Camk2a-Cre;Rng105 f/+ ). ****p<0.001 (genotype effect) in the trial 3 using two-way repeated measures ANOVA and one-way ANOVA. ( B ) Rotarod test. Longest retention time on the rod (left) and the number of falls from the rod (right) during a 3 min trial on 3 consecutive days. n = 12 ( Rng105 f/f ), 12 ( Camk2a-Cre;Rng105 f/f ) and 6 ( Camk2a-Cre;Rng105 f/+ ). In A and B, ## p<0.01, #### p<0.001 (trial effect) using one-way repeated measures ANOVA for each genotype. ‘#” is colored corresponding to the genotype. Data are represented as the mean ± s.e.m. See also .

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques:

Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: ( A ) Light/dark transition test. Left, distance traveled in the light ( L ) and dark ( D ) chambers; middle, time spent in the light chamber; right, the number of transition during a 5 min trial. n = 13 ( Rng105 f/f ), 14 ( Camk2a-Cre;Rng105 f/f ) and 13 ( Camk2a-Cre;Rng105 f/+ ). No significant differences were detected among the genotypes using two-way repeated measures ANOVA or one-way ANOVA. ( B ) Novel object recognition test. The number of interactions with the indicated objects during a 5 min exploration. Object two was replaced by a novel object 3 in the 2nd session. n = 6 ( Rng105 f/f ) and 4 ( Camk2a-Cre;Rng105 f/f ). *p<0.05 using two-way repeated measures ANOVA followed by paired t-test. Data are represented by dot plots with the mean.

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques:

Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: (A−D) Morris water maze. Escape latency to the platform in visible ( A ) and hidden ( B ) platform tasks. Density plot of swim paths averaged from all mice ( C ) and the time spent in each quadrant ( D ) during the probe test. In C, occupancy time is indicated by blue (low) to yellow (high) gradient. Red circles in C, platform location; T, target; R, right; L, left; O, opposite quadrants. n = 22 ( Rng105 f/f ), 18 ( Camk2a-Cre;Rng105 f/f ) and 16 ( Camk2a-Cre;Rng105 f/+ ). In A and B, ****p<0.001 (genotype effect) using two-way repeated measures ANOVA; ### p<0.005, #### p<0.001 (trial effect) using two-way and one-way repeated measures ANOVA for each genotype. In D, *p<0.05, **p<0.01, ***p<0.005 (genotype effect) using two-way repeated measures ANOVA and one-way ANOVA followed by Tukey-Kramer test; ## p<0.01, #### p<0.001 (quadrant effect) using two-way and one-way repeated measures ANOVA followed by Bonferroni post-hoc paired t-test. ( E ) Contextual fear conditioning test. Time spent in the dark chamber before foot shock (Pre-FS) and at 5 min, 1 day and 1 week after the conditioning. n = 16 ( Rng105 f/f ), 16 ( Camk2a-Cre;Rng105 f/f ) and 14 ( Camk2a-Cre;Rng105 f/+ ). *p<0.05, ****p<0.001 (genotype effect) using two-way repeated measures ANOVA and one-way ANOVA followed by Tukey-Kramer test; ## p<0.01, #### p<0.001 (trial effect) using two-way and one-way repeated measures ANOVA followed by Bonferroni post-hoc paired t-test. ‘#” is colored corresponding to the genotype. Data are represented as the mean ± s.e.m. In D and E, individual values are plotted by dots. See also .

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques:

Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: Freezing time in a test chamber, in which the mouse had received foot shock 5 days before. n = 8 ( Rng105 f/f ) and 4 ( Camk2a-Cre;Rng105 f/f ). ****p<0.001 (genotype effect) using two-way repeated measures ANOVA followed by Student's t-test. #### p<0.001 (context effect) using two-way repeated measures ANOVA followed by paired t-test. Data are represented as the mean ± s.e.m. with dot plots of individual values.

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques:

Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: ( A ) Schematic diagram of a mouse hippocampal slice. ( B ) A hippocampal slice from an adult mouse (P12 weeks) before (left) and after (right) isolation of CA1 SR. Nuclei are stained with Yo-Pro1 (green). SP, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunosum-moleculare. ( C ) Isolated SP (top) and SR (bottom). Scale bars in B and C, 500 µm. ( D ) An MA plot of mRNAs showing their relative enrichment in dendrites (SR) vs. soma (SP) in control ( Rng105 f/f ) mice. D- and S-mRNAs were identified statistically from three independent experiments with RNA-seq analysis. DAI, dendritic accumulation index.

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques: Isolation, Staining, RNA Sequencing Assay

Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: ( A ) MA plots of mRNAs in the hippocampal CA1 of Rng105 f/f mice (left) and Camk2a-Cre;Rng105 f/f mice (right). ( B ) Heat maps showing DAI of each mRNA in Rng105 f/f mice (left lane), in Camk2a-Cre;Rng105 f/f mice (middle lane), and relative DAI of each mRNA in Camk2a-Cre;Rng105 f/f mice compared with Rng105 f/f mice (right lane). ( C ) Relationship between somato-dendritic localization and RNG105 conditional deletion-dependent localization changes of D- and S-mRNAs. ( D ) Gene ontology enrichment analysis of D-mRNAs (red) and S-mRNAs (blue). ( E ) Gene ontology enrichment analysis of D-mRNAs whose localization to dendrites was reduced in Camk2a-Cre;Rng105 f/f mice (the relative DAI was below 0.8). The numbers in D and E indicate Benjamini values.

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques:

Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: ( A and B ) MA plots of mRNAs classified in ‘regulation of Arf protein signal transduction’ ( A ) and ‘structural constituent of ribosome’ ( B ) in Rng105 f/f mice (left) and Camk2a-Cre;Rng105 f/f mice (right). Gray, all mRNAs; red, D-mRNAs; blue, S-mRNAs. D-mRNAs classified in ‘regulation of Arf protein signal transduction’ (▲) and ‘structural constituent of ribosome’ (●) are accentuated. DAI, dendritic accumulation index.

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques: Transduction

Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: ( A and B ) Immunostaining for GluR1 ( A ) and GluR2 ( B ) in cultured neurons (9 DIV) from the cerebral cortex of E17.5 Rng105 +/+ and Rng105 −/− littermates. The neurons were cultured with (+) or without (-) TTX and APV prior to the staining. GluR1 and GluR2 staining before permeabilization (green, surface proteins), after permeabilization (magenta, intracellular and residual surface proteins), and merged images (total proteins) are shown. GluR1 and GluR2 are distributed in a punctate manner both in the soma and dendrites. The insets show magnified images of boxed areas. Arrowheads denote representative GluR1 and GluR2 puncta which were stained both before and after permeabilization (white), only before permeabilization (yellow) and only after permeabilization (blue). Scale bars, 10 µm. ( C and D ) Quantitative analysis of GluR1 and GluR2 surface expression in dendrites. C, the number of surface GluR1 puncta in dendrites normalized by the number of total GluR1 puncta (left), and fluorescence intensity of surface GluR1 puncta in dendrites normalized by GluR1 fluorescence intensity after permeabilization and in the soma (right). D, the same quantification for GluR2. Data are represented as the mean ± s.e.m. In C, n = 31 ( Rng105 +/+ , −), 35 ( Rng105 +/+ , +), 34 ( Rng105 −/− , −), and 33 ( Rng105 −/− , +) neurons from 4 experiments. In D, n = 39 ( Rng105 +/+ , −), 40 ( Rng105 +/+ , +), 39 ( Rng105 −/− , −), and 38 ( Rng105 −/− , +) neurons from 4 experiments. ***p<0.005, ****p<0.001 using two-way ANOVA followed by post-hoc Student's t-test. See also .

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques: Immunostaining, Cell Culture, Staining, Expressing, Fluorescence

Journal: eLife

Article Title: RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation

doi: 10.7554/eLife.29677

Figure Lengend Snippet: ( A ) Total cell lysates of surface-biotinylated primary cultured neurons (9 DIV) from E17.5 wild-type mouse cerebral cortex (total), and avidin agarose beads-bound fractions of the lysates (surface), were immunoblotted with the anti-GluR1 antibody. Control neurons were mock-treated without biotin. Arrow and arrowhead indicate biotin-labeled surface GluR1 and non-labeled intracellular GluR1, respectively. Lanes were cut and moved horizontally in the same membrane. ( B and C ) TTX/APV-treated and untreated primary cultured neurons (9 DIV) from Rng105 +/+ and Rng105 −/− littermates (E17.5) were surface biotinylated and analyzed as in A. Immunoblotting for GluR1 ( B ) and GluR2 ( C ). Arrows and arrowheads indicate biotin-labeled surface GluR1/2 and non-labeled intracellular GluR1/2, respectively. In the bottom panel in B, twice the amount of samples from Rng105 −/− neurons were loaded, which showed more clearly that the ratio of surface/intracellular GluR1 was lower in Rng105 −/− neurons than in Rng105 +/+ neurons. In A−C, numbers on the left indicate molecular mass (kDa). ( D and E ) Quantitative analysis of the ratio of surface/intracellular GluR1 and GluR2 in the biotinylation assay. The intensity of upper GluR1/2 bands in the avidin beads-bound fraction (arrows in B and C) and lower GluR1/2 bands in the total lysate (arrowheads in B and C) was measured and the surface/intracellular ratio was calculated. D, GluR1; E, GluR2. Data are represented as the mean ± s.e.m. n = 9 from 3 littermates each of Rng105 +/+ and Rng105 −/− mice. *p<0.05 using two-way ANOVA followed by post-hoc Student's t-test. Attached Files.

Article Snippet: The extracts were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) and probed with an anti-RNG105 polyclonal antibody , anti-α-tubulin monoclonal antibody (1:2,000, DM1A, Sigma-Aldrich, St. Louis, MO, USA), or anti-GFP monoclonal antibody (1:500, GF200, Nacalai Tesque, Kyoto, Japan).

Techniques: Cell Culture, Avidin-Biotin Assay, Labeling, Western Blot, Cell Surface Biotinylation Assay

Journal: Journal of Biological Chemistry

Article Title: Anticancer Drugs Up-regulate HspBP1 and Thereby Antagonize the Prosurvival Function of Hsp70 in Tumor Cells

doi: 10.1074/jbc.m707547200

Figure Lengend Snippet: FIGURE 1. Association of HspBP1 with Hsp70 in cells. A, lysates (200 g of protein) of HeLa S3 cells were subjected to immunoprecipitation (IP) with antibodies to HspBP1, Hsp70, or Hsc70 or with nonimmune immunoglobulin G. The resulting precipitates, as well as the original cell lysates (40 g of pro- tein),werethensubjectedtoimmunoblotanalysis(IB)withthesameantibod- ies, as indicated. Arrowheads and asterisks indicate specific and nonspecific bands, respectively. B, HeLa S3 cells were transfected for 24 h with expression plasmidsforEGFP-taggedHspBP1oritsdeletionmutants(shownintheupper panel). Cell lysates (200 g of protein) were subjected to immunoprecipita- tion with anti-Hsp70, and the resulting precipitates were subjected to immu- noblot analysis with antibodies to Hsp70 or GFP (lower left panel). Cell lysates (40 g of protein) were also subjected directly to immunoblot analysis with the same antibodies (lower right panel). All data are representative of three separate experiments.

Article Snippet: Other antibodies used included those to -actin (A4700) from Sigma, Bid (number 2002) or to caspase-3 cleaved at Asp175 (number 9661) from Cell Signaling Technology (Beverly, MA), cathepsin L (clone 22) from BD Biosciences (Bedford, MA), cathepsin B (CA10) from Calbiochem (La Jolla, CA), green fluorescent protein (GF200) from Nacalai Tesque (Kyoto, Japan), Hsp70 (sc-24) from Santa Cruz Biotechnology (Santa Cruz, CA), and Hsc70 (SPA-815) from Stressgen Bioreagents (Victoria, British Columbia, Canada).

Techniques: Immunoprecipitation, Transfection, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Anticancer Drugs Up-regulate HspBP1 and Thereby Antagonize the Prosurvival Function of Hsp70 in Tumor Cells

doi: 10.1074/jbc.m707547200

Figure Lengend Snippet: FIGURE 5. Up-regulation of HspBP1 in tumor cells by treatment with anticancer drugs. HeLa S3 or MKN1 cells were incubated for the indicated times in the absence (Cont) or presence of 100 nM vincristine, 100 nM paclitaxel, or 34 M etoposide, after which cell lysates (40 or 20 g of protein for HeLa S3 and MKN1 cells, respectively) were subjected to immunoblot analysis with antibodies to Hsp70, HspBP1, or -actin. Asterisks indicate nonspecific bands. Data are representative of three separate experiments (lower panels). The intensity ofHspBP1bandswasdeterminedbydensitometryandexpressedrelativetothevalueforcontrolcells.Dataare mean S.D. of values from three separate experiments (upper panel). *, p 0.05; **, p 0.01 versus corre- sponding value for control cells.

Article Snippet: Other antibodies used included those to -actin (A4700) from Sigma, Bid (number 2002) or to caspase-3 cleaved at Asp175 (number 9661) from Cell Signaling Technology (Beverly, MA), cathepsin L (clone 22) from BD Biosciences (Bedford, MA), cathepsin B (CA10) from Calbiochem (La Jolla, CA), green fluorescent protein (GF200) from Nacalai Tesque (Kyoto, Japan), Hsp70 (sc-24) from Santa Cruz Biotechnology (Santa Cruz, CA), and Hsc70 (SPA-815) from Stressgen Bioreagents (Victoria, British Columbia, Canada).

Techniques: Incubation, Western Blot, Control