Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Schematic diagram depicting different types of alternatively spliced events (left). The bar graph (right) shows the distribution of alternatively spliced events among those that are differentially spliced in total thymus in BRD4 knock-out versus wild type (WT) (FDR < 0.05). Comparison of splicing events between WT and BRD4-deficient cells was based on transcripts expressed in both. (B) Developmental stages in thymocyte differentiation, DN (CD4, CD8 DN), ISP (CD8+ ISP), DP (CD4, CD8 DP), CD4, and CD8 single-positive thymocytes are shown. Arched arrows denote level of proliferative activity in DN and ISP thymocytes. (C) Bar graph showing the total number of differentially spliced events in the different thymocyte subpopulations in BRD4 knock-out versus wild-type thymus (FDR < 0.05), derived from RNA-seq analysis. BRD4 was conditionally deleted in DN thymocytes by LCK-Cre (Gegonne et al., 2018). Comparison of splicing events between WT and BRD4-deficient cells was based on transcripts expressed in both. See also Figure S1.

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Knock-Out, Comparison, Activity Assay, Derivative Assay, RNA Sequencing

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: Summary of Immune Relevant Genes Whose Patterns of Splicing Are Altered by BRD4 Deficiency and the Thymocyte Subset in which the Alteration Occurs

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Binding Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: RNA from the different thymocyte subpopulations was subjected to RT-PCR for the indicated genes: CD45 (A and B), Arhgef1 (C and D), and Picalm (E and F). (A, C, and E) Upper panels: schematic diagrams depicting partial gene structure of the alternatively spliced genes CD45 (A), Arhgef1 (C), and Picalm (E). Rectangular boxes represent the exons, and the horizontal straight lines connecting the boxes represent the introns; the numbers below the boxes refer to the exon number of the gene, and numbers inside the boxes refer to the length of the exons; the numbers within the terminal exons do not refer to the actual exon length but the length amplifiable by the RT-PCR primers. The arrow heads show the approximate positions of the RT-PCR primers; boxes with hashed lines show the alternative exons; and curved lines connecting the boxes depict the splicing pattern. WT and KO refer to the splicing pattern prevalent in either the wild-type or knock-out thymocytes as determined by RNA-seq analysis. Lower panels: ethidium bromide stained agarose gels showing RT-PCR products derived from total RNA from BRD4 WT and KO thymocytes. (B, D, and F) Bar graphs of the RT-PCR results for CD45 (B), Arhgef1 (D), and Picalm (F). The ratios A/A+B (ratio of included exon transcript/total transcripts) were used as measure of alternative splicing and represent the average of three separate RT-PCR analyses. #, p < 0.05, significant difference between WT subpopulations, relative to WT DN; *p < 0.05, significant difference between WT and KO for the specific subpopulation. See also Figure S2.

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Reverse Transcription Polymerase Chain Reaction, Knock-Out, RNA Sequencing, Staining, Derivative Assay, Alternative Splicing

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Effect of JQ1 or dBET6 treatment on the binding of BRD4 across the gene. ALL, TSS+gene body+TTS+intergenic; TSS, transcription start site; Gene body, between TSS and TTS; TTS, transcription termination site; intergenic, all remaining sequences. The peak distribution, in the absence of treatment is as follows: TSS, 1123; gene body, 4065; TTS, 226; intergenic, 1827. (B) Bar graph showing the distribution of alternative splice events among the differentially spliced events in response to JQ1 treatment or dBET6 treatment in T-ALL cells. (C) Bar graph showing the fraction of alternative splice (AS) genes that also have BRD4 associated with them at the TSS (pkAS). The total number of BRD4 peaks detected at the TSS across the genome was 1123. (D) Bar graph showing the fraction of AS genes that are also differentially expressed (DE) in response to JQ1 or dBET6 treatment. p values for (C) and (D) were obtained using a hypergeometric test, which tests the probability that the frequency of AS genes derived from either DE genes (overlap) or genes with BRD4-bound TSS peaks is larger than expected from the population; a low p value suggests the enrichment of AS genes in either DE genes or genes with BRD4 TSS peaks. See also Figures S3 and S4.

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Binding Assay, Derivative Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Immunoblot of BRD4 immunoprecipitates from thymocyte nuclear extracts (with and without benzonase treatment) with indicated antibodies to splicing factors FUS, HnRNPL, and U1–70. The immunoprecipitates from a single extract were run on either a 6% gel to visualize BRD4 and Fus or on a 10% gel to visualize HnRNPL and U1–70. The values under the IP lanes indicate the enrichment of anti-BRD4 co-IP, relative to the IgG control. (B, left) Immunoblot of BRD4 immunoprecipitates from HeLa nuclear extracts with indicated antibodies to splicing factors FUS, HnRNPM, U1–70, and U1-A. (B, right) Immunoblot of FUS immunoprecipitates from HeLa nuclear extracts with indicated antibodies to BRD4 and splicing factors HnRNPM, U1–70, and U1-A. (C) Schematic representation of BRD4 and BRD4-deletion mutants. The coordinates of the mouse BRD4 mutations are as follows. WT BRD4, 1402 aa; DN, 722–1402 aa; ΔC, 1–699aa; ΔBD1, 146–1402 aa; ΔBD2+B, 1–349/599–1402 aa; ΔB, 1–502/549–1402 aa; ΔET, 1–600/684–1402 aa; ΔHAT, 1–1156/1198–1402 aa. (D) Immunoblots showing pull-down analysis of recombinant BRD4 with recombinant HnRNPM. rHnRNPM (0.25 μg) was pulled down with rflag-BRD4 (0.5 μg) immobilized on Flag beads. Immunoblots were with anti-HnRNPM (upper) and anti-BRD4 (lower). (E) Immunoblots showing pull-down analysis of recombinant BRD4 with recombinant FUS. rFUS (0.25 μg) was pulled down with rflag-BRD4 (0.5 μg) WT or equimolar amounts of N-terminal or C-terminal BRD4 truncation mutants immobilized on Flag beads. Immunoblots were with anti-FUS (upper) and anti-BRD4 (lower). (F) Binding of HnRNPM (left panel) and FUS (right panel) to BRD4 mutants was assessed in pull-down assays with rBRD4 immobilized on Flag beads and immunoblotting with appropriate antibodies. The results represent the average of two experiments. (G) Retention of FUS and HnRNPM to BRD4 mutants, relative to the WT, was quantified as the fraction of input and normalized to the extent of binding to BRD4 WT. All results are representative of at least two independent experiments. See also Figure S5.

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Control, Recombinant, Binding Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Proximity ligation assays (PLAs) were performed on primary thymocytes with anti-BRD4 and the antibodies for the indicated splicing factors. The PLAs are all significantly above the single antibody controls (Figure S6C). (B) PLA was performed using anti-BRD4 and the antibodies for the indicated splicing factors on fixed HeLa cells that had been treated with JQ1 (500 nM)/ DMSO for 6 hr. There is no significant difference (p > 0.05) between the treated and control PLA samples for either HnRNPM or Fus; both PLAs are significantly above single antibody alone controls (Figure S6C). PLA interaction is shown in red; DAPI staining in blue. See also Figures S6C and S6D.

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Ligation, Control, Staining

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Metagene profile of BRD4 and FUS CHIP datasets showing colocalization of BRD4 and FUS at the TSS. (B) Log2 enrichment of reads in genomic features along the metagene body. (C) Enrichment heatmap showing co-localization of BRD4 with FUS across the genome. (D) Genome browser views of DNAAF3, ROBO3, and MAN1A1, showing BRD4 and FUS co-localization around the TSS and gene body. See also Figures S6A and S6B.

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques:

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti- BRD4 [Epitope BRD4 C-terminal peptide (CFQSDLLSIFEENLF)] , Covance (Cutom antibody synthesis); Dey et al., 2000 , N/A.

Techniques: Recombinant, In Situ, cDNA Synthesis, Plasmid Preparation, Mutagenesis, Clone Assay, Software, Sequencing, Alternative Splicing

Journal: PLoS ONE

Article Title: Fn3 proteins engineered to recognize tumor biomarker mesothelin internalize upon binding

doi: 10.1371/journal.pone.0197029

Figure Lengend Snippet: We started with a naïve yeast surface display library with 2.8 x 10 9 variants of the Fn3 non-antibody scaffold. The library was sorted for full-length protein expression, detected by an antibody to a terminal c-myc epitope tag, and binding to MSLN using MACS and FACS. Red polygon indicates example cell population collected for further enrichment and analysis. Additional diversity was introduced into the enriched library through a single round of mutagenic PCR and sorting of this second generation library resulted in further enrichment for MSLN binding variants. A double-negative population of yeast cells is characteristic of yeast surface display.

Article Snippet: The magnetic sorts were followed by a fluorescent-activated cell sorting (FACS) selection for full-length clones using an antibody against the C-terminal c-myc epitope tag (clone 9E10, Life Technologies, 1:50) and a goat anti-mouse phycoerythrin (PE) conjugate (Sigma #P9670, 1:25).

Techniques: Expressing, Binding Assay

Journal: bioRxiv

Article Title: The costimulatory domain influences CD19 CAR-T cell resistance development in B-cell malignancies

doi: 10.1101/2025.02.28.640707

Figure Lengend Snippet: ( A ) Scheme of procedure used for generation of resistant models (left panel). Lymphoma (Raji) and B-ALL (Nalm-6) models were established through 19 subsequent and parallel expositions to effectors (unmodified T-cells, CD19-4-1BB- or CD19-CD28 CAR-T cells), in the same conditions and E:T ratio for every passage. The structures of CAR-CD19 constructs bearing 4-1BB or CD28 costimulatory domain (right panel) were identical to clinically used product tisa-cel and axi-cel, respectively. ( B ) Efficacy of T-cells transduction with CAR-CD19-4-1BB and CAR-CD19-CD28 constructs. Detection of CAR-positive T-cells (a representative plot for one of the PBMCs’ donors) was performed by flow cytometry staining with RTX followed by secondary AlexaFluor 647-conjugated anti-Fc anibody (left) or anti-scFv (FMC63) antibody (right). ( C ) Flow cytometry analysis of CD19 surface expression in Raji cells (left panel) and Nalm-6 cells (right panel) during the process of long-term co-culture with CD19 CAR-T cells bearing either 4-1BB or CD28 costimulatory domain. The CD19 expression was detected by the HIB19 antibody clone after targets-effectors co-culture separation and targets recovery (up to 10 days). Raji or Nalm-6 cells co-cultured with unmodified T-cells are shown in black (con), co-cultured with CD19-4-1BB CAR-T cells in blue (Raji/Nalm-6 CAR-4-1BB) and with CD19-CD28 CAR-T cells in magenta (Raji/Nalm-6 CAR-CD28). The staining of anti-CD19 antibody (filled histograms) was compared to isotype controls (transparent histograms). p11-p19 reffers to the passage number. ( D ) Flow cytometry-based analysis of sensitivity of Raji (upper panel) and Nalm-6 (lower panel) cells to unmodified T-cells, CD19-4-1BB CAR-T cells or CD19-CD28 CAR-T cells. In the experiment, control (con) Raji and Nalm-6 target cells and their counterparts after long-term co-culture (19 passages) with CD19 CAR-T cells incorporating 4-1BB or CD28 domain were used. The plot shows the % cytotoxicity of effectors against targets. Data are presented as the mean ± SD from 5-7 donors analyzed in 2 technical repeats each. Statistical analysis was performed using two-way ANOVA with interaction and Tukey’s post-hoc test for multiple comparisons. Only selected significances were shown on the plots.

Article Snippet: Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-CD19 recognizing epitope surrounding Leu427 (Clone 1; 1:1000 dilution, Cell Signaling Technology, Cat #90176, RRID: AB_2800152), anti-CD19 recognizing C-terminus residues (Clone 2; 1:1000 dilution, Cell Signaling Technology, Cat #3574, RRID: AB_2275523) or anti-tubulin (1:1000, Sigma-Aldrich, Cat #CP06, RRID: AB_2617116).

Techniques: Construct, Transduction, Flow Cytometry, Staining, Expressing, Co-Culture Assay, Cell Culture, Control

Journal: bioRxiv

Article Title: The costimulatory domain influences CD19 CAR-T cell resistance development in B-cell malignancies

doi: 10.1101/2025.02.28.640707

Figure Lengend Snippet: ( A-B) Flow cytometry analysis of CD19 surface level with different anti-CD19 antibodies clones – HIB19 (black), FMC63 (orange), SJ25C1 (turquoise) and J3-119 (red) in Raji and Nalm-6 control (con, black) and after long-term co-culture (19 passages) with effectors - CD19-4-1BB CAR-T ( A , blue) or CD19-CD28 CAR-T cells ( B , magenta). The staining of anti-CD19 antibody (filled histograms) was compared to isotype controls (transparent histograms). ( C) The stability of resistance phenotype on Raji and Nalm-6 CAR-4-1BB and CAR-CD28 models was evaluated by flow cytometry CD19 surface level analysis with HIB19 clone (left panel) and FMC63 clone (right panel) after a culture of targets without effectors (8, 15, 22 and 29 days starting from day 0 – establishment of the models. ( D, F) Flow cytometry-based analysis of CD19 intracellular epitope (clone D4V4B recognizing residues surrounding Leu427) level in Raji ( D ) and Nalm-6 ( F ) WT, control, CAR-4-1BB and CAR-CD28 tumor cells. Data show the representative experiment from 2 biological replicates. ( E, G) Western blotting analysis of total CD19 protein level in Raji ( E ) and Nalm-6 ( G ) WT, control, CAR-4-1BB, and CAR-CD28 tumor cells evaluated by two antibodies recognizing different CD19 intracellular epitopes – residues surrounding Leu427 (clone D4V4B) or C-terminus residues (polyclonal antibody). Tubulin (TUB) was used as a loading control.

Article Snippet: Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-CD19 recognizing epitope surrounding Leu427 (Clone 1; 1:1000 dilution, Cell Signaling Technology, Cat #90176, RRID: AB_2800152), anti-CD19 recognizing C-terminus residues (Clone 2; 1:1000 dilution, Cell Signaling Technology, Cat #3574, RRID: AB_2275523) or anti-tubulin (1:1000, Sigma-Aldrich, Cat #CP06, RRID: AB_2617116).

Techniques: Flow Cytometry, Clone Assay, Control, Co-Culture Assay, Staining, Western Blot

Journal: bioRxiv

Article Title: The costimulatory domain influences CD19 CAR-T cell resistance development in B-cell malignancies

doi: 10.1101/2025.02.28.640707

Figure Lengend Snippet: ( A) Scheme of CD19 gene fragment (exon 1 to exon 7; grey blocks) together with the location of the primers used for RT-qPCR experiments (ex1-2, ex3-4, ex4-5, black arrows) and codons coding amino acid residues (W159, R163, P222, K220) essential for FMC63 epitope recognition. ( B-C) RT-qPCR experiments showing relative CD19 mRNA level in Raji ( B ) and Nalm-6 ( C ) control (con) cells and after 19 passages of effectors’ exposures to CD19-4-1BB-CAR-T (4-1BB, blue) or CD19-CD28-CAR-T-cells (CD28, magenta). The relative mRNA expression was assessed by 3 different pairs of CD19 -specific primers covering exon 1-2, exon 3-4 or exon 4-5 junctions. The plot shows the CD19 mRNA level calculated with ΔCt method relative to the mean of TBP and GUSB as housekeeping genes. Data are presented as the mean ± SD from 3-4 biological repeats performed in technical triplicates. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test for multiple comparisons. ( D) RNAseq analysis of CD19 transcript level in Raji and Nalm-6 control (con) and after exposition (19 passages) to CD19-4-1BB CAR-T cells (4-1BB) or CD19-CD28 CAR-T cells (CD28). The plot (left panel) shows raw expression (transcript per million, TPM) of CD19 transcript. Data is presented as mean ± SD from 3 biological replicates. Right panel shows the results of differential gene expression analysis (DESeq2) – the log 2 FC (fold change) and the p-value between control and 4-1BB or CD28 variants in Raji and Nalm-6 cell lines. ( E-F) CD19 sequence in Raji ( E ) and Nalm-6 ( F ) after exposition (19 passages) to CD19-4-1BB CAR-T cells (4-1BB) or CD19-CD28 CAR-T cells (CD28), as detected in RNAseq data and compared with CD19 RefSeq. The scheme shows the fragments of nucleotide sequences (blue – C, cytosine; orange – G, guanine; red – T, tymine; green – A, adenine) together with protein sequences, amino acid positions and exon numbers across the reference CD19 gene and those detected in Raji and Nalm-6 CAR-4-1BB/CD28 variants. The results of mutations detected in 4-1BB cell lines (amino acids and together with variant allele frequency, VAF [%]) are marked in blue. ( G-H) Intron 2 ( G ) or intron 6 ( H ) retention analysis in Raji and Nalm-6 control cells and after exposition (19 passages) to CD19-4-1BB CAR-T cells (4-1BB) or CD19-CD28 CAR-T cells (CD28) based on RNAseq data. Left panels show sashimi plots with coverage and the number of junction reads in Raji (upper panel) and Nalm-6 (lower panel) from pooled data of 3 biological replicates of each variant. The plots (right panels) show the frequency (% of all isoforms) of intron 2 ( F ) or intron 6 ( G ) retention calculated using the R/Bioconductor package ASpli. The data are presented as mean ± SD from 3 biological replicates. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test for multiple comparisons. Only selected significances were shown on the plots.

Article Snippet: Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-CD19 recognizing epitope surrounding Leu427 (Clone 1; 1:1000 dilution, Cell Signaling Technology, Cat #90176, RRID: AB_2800152), anti-CD19 recognizing C-terminus residues (Clone 2; 1:1000 dilution, Cell Signaling Technology, Cat #3574, RRID: AB_2275523) or anti-tubulin (1:1000, Sigma-Aldrich, Cat #CP06, RRID: AB_2617116).

Techniques: Quantitative RT-PCR, Control, Expressing, Gene Expression, Sequencing, Variant Assay

Journal: bioRxiv

Article Title: The costimulatory domain influences CD19 CAR-T cell resistance development in B-cell malignancies

doi: 10.1101/2025.02.28.640707

Figure Lengend Snippet: ( A-B ) Venn diagrams showing the comparison of significantly upregulated or downregulated genes between Raji CAR-4-1BB and CAR-CD28 ( A, upper panel ), and Nalm-6 CAR-4-1BB and CAR-CD28 ( B, upper panel ). The significantly changed genes (p-value > 0.05, log 2 FC > 1 (up) or log 2 FC < −1 (down)) were extracted based on differential gene expression analysis (DESeq2). Volcano plots showing the results of differential gene expression analysis (DESeq2) between Raji control and CAR-4-1BB ( A, middle panel ), Raji control and CAR-CD28 ( A, lower panel ), Nalm-6 control and CAR-4-1BB ( B, middle panel ) and Nalm-6 control and CAR-CD28 ( B, lower panel ). The cut-off point for p-value was set as 0.05 and for log 2 FC as 1 or −1. ( C-D) Venn diagrams showing the comparison of significantly upregulated ( C ) or downregulated ( D ) genes between B-ALL patients treated with tisa-cel (re-analysis of RNAseq data from Orlando et al. (2018) and, Raji and Nalm-6 after long-term co-culture with CD19-4-1BB CAR-T cells (CAR-4-1BB). The significantly changed genes (p-value > 0.05; log 2 FC > 1 (up) or log 2 FC < −1 (down)) were extracted based on differential gene expression analysis (DESeq2) between relapsed samples vs. screening samples from Orlando’s data set and CAR-4-1BB vs. control samples in Raji and Nalm-6 models.

Article Snippet: Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-CD19 recognizing epitope surrounding Leu427 (Clone 1; 1:1000 dilution, Cell Signaling Technology, Cat #90176, RRID: AB_2800152), anti-CD19 recognizing C-terminus residues (Clone 2; 1:1000 dilution, Cell Signaling Technology, Cat #3574, RRID: AB_2275523) or anti-tubulin (1:1000, Sigma-Aldrich, Cat #CP06, RRID: AB_2617116).

Techniques: Comparison, Gene Expression, Control, Co-Culture Assay

Journal: bioRxiv

Article Title: The costimulatory domain influences CD19 CAR-T cell resistance development in B-cell malignancies

doi: 10.1101/2025.02.28.640707

Figure Lengend Snippet: ( A ) General simulation algorithm presented on activity diagram with the six subsequent experimentation stages. ( B) Graphical representation on the histogram of the distinct subpopulations (resistant, low-antigen and high-antigen cells with defined antigen levels. ( C) Main parameters set for simulations. ( D) Histograms showing changes in CD19 MFI in subsequent passages (iterations) mimicking contact with CD19-4-1BB CAR-T cells (left) or CD19-CD28 CAR-T cells (right). The graphs show selected iteration from 19 that were performed.

Article Snippet: Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-CD19 recognizing epitope surrounding Leu427 (Clone 1; 1:1000 dilution, Cell Signaling Technology, Cat #90176, RRID: AB_2800152), anti-CD19 recognizing C-terminus residues (Clone 2; 1:1000 dilution, Cell Signaling Technology, Cat #3574, RRID: AB_2275523) or anti-tubulin (1:1000, Sigma-Aldrich, Cat #CP06, RRID: AB_2617116).

Techniques: Activity Assay