EMBO/sd-character-level-ner huggingface dataset:
D Up, Representative western-blot showing H2AX protein levels from whole cell extracts in HCC70 TN BC cell line transfected with non-targeting siRNA (siCtrl) or an siRNA directed against H2AX (siH2AX). Actin is used as internal control for protein loading. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above).
A WT and E109AE110A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, cells were untreated or treated with TSQ for 6 hrs (please note that the same WT control was used in Fig. EV3A). Cell death was measured by PI staining based on flow cytometry. Data are plotted as mean ± SEM of three independent experiments. B Cellular fractions from (A) were analysed by Western blot from BN-PAGE or SDS-PAGE using antibodies as indicated. The same WT control was used in Fig. 4A. Representative of three independent experiments. C Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.
(A) Isolation of the STC elicited by the last pulse of a high-frequency train of action potentials. Left: superimposed representative traces of the inward current evoked in an astrocyte (held at -90 mV) by extracellular stimulation with a train of 10 pulses (trace a: black) and a train of 9 pulses (trace b: brown) at 50 Hz in a WT cortical slice. The inward current elicited by the 10th pulse was obtained by subtracting the current elicited by 9 pulses from that elicited by 10 pulses (trace a-b). Right: The STC elicited by the 10th pulse (trace a-b-c) was obtained by subtracting the exponential function that simulates the TBOA-insensitive current elicited by a single pulse (trace c, obtained as in Figure 1C) to the 10-9 pulses difference current (trace a-b). The decay of the STC elicited by the 10th pulse was best fitted by a single exponential function with τdecay = 8.04 ms (in red).
Complementation of α1-tubulin(-) parasites with different α1-tubulin constructs lacking introns (α1∆introns) or featuring only codon-modified exons (α1cm&∆introns). Scanning electron micrographs show the aberrant shape of the α1cm&∆introns-expressing sporozoites. See also Fig EV5. Scale bar: 1 μm.
F-I Number of HSC clones that produce different amounts of Gr, B, CD4 T, and CD8 T cells. We combined the sequencing data with the flow cytometry data to calculate clonal abundance for each clone as follows: Clonal abundance =100%*(Each cell population (Gr, B, CD4T or CD8T cells) % WBCs) * (Donor % Each cell population) * (GFP % Donor cells) * (number of reads for each barcode) / (total reads of all barcodes Data information: Data were collected at month 4 after secondary transplantation and presented as mean ± SEM. n=7 mice for the primary control (WT+WT) and deficient co-transplantation (WT + uMT-/-) groups. n=4 mice for the secondary transplantation control group (WT + WT) and n=6 for the secondary deficient co-transplantation group (WT + uMT-/-). *p<0.05 and **p< 0.01 (two-tailed and two-sample equal variance Student"s t-test)
E. Native gel showing the inability of CENP-Cmotif to compete out CENP-CCR bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome. Lane 2 and 6: CENP-CCR/CENP-A nucleosome complex. Lanes 3-5: Increasing amounts of CENP-Cmotif are added to the pre-formed CENP-A/CENP-CCR complex. Formation of smear at high amounts of CENP-Cmotif added, indicates that CENP-Cmotif, at high concentrations, non-specifically binds CENP-A/CENP-CCR complex rather than replacing bound CENP-CCR. Lane 7-9: Increasing amounts of CENP-CCR-short are added to the pre-formed CENP-A/CENP-CCR complex. Formation of bands with higher mobility indicates that smaller CENP-CCR-short is effectively replacing bigger CENP-CCR bound to CENP-A nucleosome.
B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. Forty-eight hours after infection, total RNA was subjected to analyzing the levels of lncRNA-MIF and lncRNA-MIF-AS by real-time RT-PCR. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).
(E) Fraction of phagocytosed GFP+ MM cells after 16 hours treatment with 10 nM CXCL12. The numbers above the columns indicate the phagocytosed/non-phagocytosed MM. Statistics: χ2 test, phagocytosed vs non phagocytosed tumor cells.
(E) Representative immunofluorescence images showing GnRH neurons migrating along Nrp1-immunoreactive vomeronasal/terminal nerve fibers (empty arrowheads) at the nose-brain junction in sagittal slices from E14.5 control and mutant embryos. GnRH neurons (green) themselves express Nrp1 (red, white arrows) in control Nrp1loxP/loxP mice whereas Nrp1 is undetectable in GnRH neurons from mutant Gnrh::cre; Nrp1loxP/loxP littermates (black arrows). Scale bar: 50μm.
(B) Cell lysates prepared from HeLa cells were subjected to subcellular fractionation by centrifugation at 100 000 g for 1 h. The membrane (lanes 1 and 3) and supernatant (lanes 2 and 4) fractions were subjected to SDS-PAGE, followed by immunoblotting with anti‐PtdIns 3‐kinase (lanes 1 and 2) and anti‐Beclin (lanes 3 and 4) antibodies.
A. Western blot analysis of the IFT22RNAi+GFP::IFT22A86R cell line probed with the anti-IFT22 antibody (bottom) and with an anti-BiP as loading control (top).
(F) Representative images (and details from boxed regions) of WT MEFs cells transfected with GFPPINK1 (green) and treated with FCCP (10 μM) to induce mitophagy. Cells were immunostained with anti-Tom20 (magenta) to reveal the mitochondrial network. Each row represents one of the three categories of GFP signal used to describe PINK1 translocation to mitochondria after mitochondrial damage (cytoplasmic, intermediate and mitochondrial). The scoring of these categories was used to describe PINK1 translocation to mitochondria in WT and MiroDKO cells at different time-points shortly after mitochondrial insult (G). Data collected from 3 independent experiments (n=3). Error bars represent s.e.m. Significance: *p<0.05 and **p<0.01
D, E Inhibition of TGF-β1 abolishes Sema3A production and NEM recruitment in vivo. Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) VEGF levels, were treated with a TGF-β1 blocking antibody. Analyses were preformed after 1 week. (D) Immunohistochemistry for Sema3A protein on frozen muscle sections with H&amp;amp;E counterstaining. The intensity of Sema3A stain was quantified as reciprocal intensity on a scale out of 250. Scale bar = 50 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: No stain, n = 10; Ctrl IgG1, n = 17; Ctrl AbαTGF-β1, n = 16; V Low IgG1, n = 23; V Low AbαTGF-β1, n = 28. **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG1 versus No stain P = 0.0069; V Low IgG1 versus Ctrl IgG1 P < 0.0001; V Low AbαTGF-β1 versus V Low IgG1 P = 0.0002.
(D) Fluctuation analysis in M. tuberculosis H37Rv. M. tuberculosis ΔcydAB was plated on 7H10 containing 100 nM Q203 (red). Parental H37Rv was plated on 7H10 with 100 nM Q203 and 6 µM ND-011992 or 2 µg/mL rifampicin (blue). The corresponding values of the median frequency of resistance is indicated in the graph.
(D) Quantification of ER-mitochondria colocalization per cell analyzed. Upon thresholding each channel, a mask for ER was generated and the mitochondria channel was superimposed on the ER mask, so the positive pixels found in both channels were quantified and referenced over the total area of the ER mask. The number of cells analyzed is indicated in white text within each column. Levels of ER-mitochondria colocalization in APP-WT cells are shown for reference as a dashed line. EV, empty vector. One-way ANOVA (n=3 independent experiments and 5-8 images/experiment; * p< 0.05).
F. Oligo#5 does not change the RNA2-editing pattern. The hypothalamic area showing oligo#5 staining was microdissected 6 hrs post injection and RNA2 was amplified and the PCR product subjected to deep sequencing. The percent of non-edited reads in total reads is shown (at least 20,000 reads per lane).
G. ST2 cells wild type or NEMO KO were stimulated with IL-17 (500 ng/ml) for indicated time points, solubilized and analyzed by immunoblotting.
F, Quantification of oligomer versus monomer ratios from Raw 264.7 cells treated with 1 μg/ml LPS, as shown in (E). Quantified results are fold change of oligomer versus monomer ratios from its corresponding vehicle treated cells. Data are presented as mean ± SD using one-way ANOVA (**)p< 0.01, from two independent experiments.
(c) Downregulation of IRGM expression by miR-196 abrogates AIEC-mediated autophagy in cells treated with autophagic inhibitors (Inh) or transfected with miR-196B and infected for 4 h with AIEC LF82 (mean of three independent experiments ± s.d.).
C. Kaplan-Meier relapse-free survival curve according to subnetwork metagenes expression levels in breast tumors. Curves were compared with a log-rank test (p-value).
K+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).
Selective Cullin-RING inhibitor MLN4924 rescued the degradation of E2F7/8 under nocodazole-arrested condition. HeLa cells were treated with DMSO, nocodazole or nocodazole plus MLN4924 (0.1 μM) for 16 hours. Cells were harvested and lysed for immunoblotting. cyclin B1 expression was used as a marker for G2 or M cell cycle progression, and γ-tubulin was used as loading control.
B. Fork symmetry in cells treated as in (A) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).
Genomic distribution of POLG DHSs. Black arrow shows a cluster upstream from POLG coding region.
A The A3B3DF complex shown with the subunits unique to the eukaryotic V1 removed, highlighting the core conserved catalytic complex labeled as "Catalytic core". The three peripheral stalks and inhibitory subunit H are found only in the eukaryotic V1, whereas A3B3DF represents the full complement of subunits in the prokaryotic V1 complex. Notably, the prokaryotic V1 is an active ATP hydrolase (Arai et al, 2009). The orientation of the A and B subunits with respect to the membrane is indicated by the arrow on the left.
G Alignment of subdomains VIb and VII of the catalytic core of the kinase domains of active (BRI1, FLS2, EFR, CRK7) and inactive RLKs (PRK4, PRK5, BIR2, SUB). Residues marked in green highlight conservation of the consensus of active protein kinases while residues highlighted in red indicate deviations from the consensus sequence. An alignment of the full kinase domains for the RLKs used in this figure can be found in Supplementary Fig S11A.
B, Immunoblot analysis of protein lysates from HeLa cells treated with 250 μM of NaAsO2 for the indicated times and run under non-reducing conditions.
except that cells were preextracted in 0.2% Triton X-100, fixed and immunostained with SUMO2/3 antibody. Representative images are shown. Scale bar, 5 μm.
GST-MICU1_EF1mut failed to pull down MICU2_WT in the absence of 2 mM Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A & H385A failed to pull-down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. C GST-MICU1_E242K failed to pull-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_D231A partly pulled-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A failed to pull down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. GST-MICU1_H385A exhibited a strong interaction with MICU2 both in the absence and presence of 2 mM Ca2+.
(A) TOM20-positive MDV formation was induced in SH-SY5Y by siRNA knockdown of Tollip. MDVs were defined as TOM20+ve/Cytochrome c (Cyt c)-ve via immunofluorescence microscopy, which indicated some colocalisation with Parkin (arrowheads).
Quantification of Ki67-positive cells in sections from SCC13 EV and SCC13 Act (clone 2) organotypic cultures (co-stained with E-cadherin to indicate epithelial cells). N=3 independent cultures, n=3 histological sections.
(A) mH2A1.2 and different neurogenesis markers are present in cortical lysates collected at various time points (E13, E15, E17, and P0).
(C) BNGE, Western blot and immunodetection, with the monoclonal (M) anti-UQCRQ antibody (Abcam ab110255), of non-transduced ∆4-CYB and WT cells. The mitoplast samples were solubilized with DDM
(D) Loss of STXBP4 diminishes the association of α-catenin with AMOT, LATS1 and YAP. HEK293A and STXBP4 KO cells were transfected with the SFB-tagged α-catenin construct and subjected to the pulldown assay.
C Boxplots of STAMP2 expression levels in normal prostate tissue, primary prostate cancer, and metastatic CRPC were analyzed in two independent GEO datasets (GSE6919 and GSE35988). The values of individual samples are presented as dots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles. The statistical significance, indicated in the tables at the bottom, was determined by Student's t-test.
Effects of compounds that target histamine receptor family members. The outline shows the compounds (listed in Dataset EV3) that target histamine receptor family members as antagonists (HRH1, HRH2, HRH3 and HRH4) or agonists. These include the compounds originally present in the Prestwick library as well as additional compounds selected from the litterature. Cells treated with this small library of compounds were analysed by automated microscopy after labeling with DAPI and antibodies against LBPA. Compounds that increased LBPA staining intensity, like thioperamide (Fig 1) or pitolisant (Fig 4B) are green, while compounds without effects are red. (n=2 independent experiments with 96 images acquired and analysed automatically).
A. Effects of selected kinase inhibitors with differential effects on Rb∆K7 vs. control MEFs from initial kinome screens. Average proliferation rate of untreated MEFs is shown on the right (white column). Arrows point to pan-CDK- and CDK2/7/9-inhibitors. Bars represent mean ±SD, n=3 independent MEFs.
(C) The complete amino acid sequences of human UBQLN1 and UBQLN4 were analyzed using the disorder/order structure prediction program PONDR-FIT. The STI-II region of UBQLN4 is indicated by the red box, and that of UBQLN1 is boxed blue. Note that the disorder tendency of the UBL, STI-I, and UBA regions is nearly identical between UBQLN1 and UBQLN4 (indicated by black and green boxes), while that of the STI-II region is quite distinct, and the STI-II of UBQLN4 shows a high probability of an ordered structure. The numbers denote the corresponding amino acid positions in these proteins.
U2OS/GFP-FAM111A cell lines that were treated or not with DOX were pre-extracted and fixed, and stained with DAPI. DAPI signal intensities were analyzed by QIBC. H. cells were stained with RPB1 antibody.
(E) I90 cells, transfected as in (D) for 24 h, were microscopically analysed for GFP‐LC3 fluorescence. Representative pictures are shown. Bar: 20 μm. Values expressed in diagram are mean±s.e.m. from three independent experiments (50 cells were counted per experiment). *P0.05 versus N1, n=3.
(c,d) Kinetic analysis of [3H]thymidine incorporation (c) and cell numbers (d) in cultures of OT-II and Irgm1−/− OT-II spleen cells stimulated with OVA peptide.
D Representative bioluminescence images (left panel) and bar plot quantification (right panel) of lung metastases 16 days after i.v. injection of HR1 gCtrl or HR1 g4Nfib cells. n = 4, means ± s.d., two tailed Student's t-test.
A, B Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and the luminal ER marker sfGFP-KDEL (green).
(H) MHCII antigen presentation in SWHEL B cells 24 hours post adoptive transfer and immunization with Ea-HEL-SRBC conjugate as detected by anti-Y-Ae surface stain.
A. Pooled SCN cells generate a combined dataset of 17,363 cells. The sequenced SCN cells carry explicit barcodes which were used to back-reference sequencing run identity. This yields a clear-cut segregation into two transcriptionally distinct cell groups. Left: 98.6% of cells in the yellow cluster are CT7 5 cells. Right: 98.8% of cells in the grey cluster are CT15.5 cells.
(a) We transfected MN-1 cells with the 4X-CLEAR luciferase reporter and with BFP-tagged TFEB or BFP empty vector, and then measured luciferase activity. Marked induction of luciferase activity occurred with BFP-TFEB; n = 3 independent experiments, ***P 0.001, t-test, WT t(4) = 48.79; ***P 0.001, t-test, AR 24Q t(4) = 22.91; ***P 0.001, t-test, AR 65Q t(4) = 14.02. Results for each line were normalized to the BFP-empty luciferase activity.
B. ΔPrPs are moderately resistant to chymotrypsin digestion. Immunoblots showing chymotrypsin-resistant fragments of Δ159 and Δ159-175 from transiently transfected N2a cell lysates digested with indicated concentrations of chymotrypsin (Chy). Note that full-length diglycoform of ΔPrP still remains at 8 µg/ml Chy (lanes 5 and 10, upper panel, closed arrowhead), whereas that of endogenous wild-type PrP is completely digested (lanes 5 and 10, lower panel, open arrowhead) and only small amounts of truncated fragments remained (curly bracket). Other smaller fragments in lane 5 and 10 (square-brackets) represent ΔPrPs.
A) Thyroid morphology was investigated using Nkx2-1 staining at E9.5 and E11.5 (sagittal sections) and at E13.5, E15.5, and E17.5 (transverse sections) in wild-type (wt) and Tubb1-/- littermates. Nkx2-1was used as a marker of early thyrocyte progenitors and differentiated thyrocytes. Delays in thyroid migration and in fusion of the median anlage to the ultimobranchial bodies were observed at E11.5 and E13.5 (arrow), respectively, in Tubb1-/- mice. At E11.5, stained migrating thyroid cells were visible along the tract only in Tubb1-/- littermates (arrow). At E17.5, a pyramidal lobe was visible in Tubb1-/- littermates (arrow). tr: trachea; ub: ultimobranchial body. Scale bar: 50 μ
(C) Representative images of DIV10 hippocampal neurons transfected for 4 days with plasmids encoding β-gal for visualizing the transfected cells and three different DCLK1 shRNAs or DCLK2 shRNA and immunostained for β-gal (red) and DCLK1 (green).
C qPCR analysis of endogenous Oct4, Nanog or endogenous Sox2expression level during reprogramming with SKO plus Flag, wild-type Gadd45a or G39A Gadd45a. (**p≤0.01; ***p≤0.001; n=3)
B. Immunoblot of phospho-S622 and pan-Drp1 in N2A cells depleted of STAT5 A and B by CRISPR/Cas9 (DKO) or with nontargeting control (NTC). Vinculin was used as a loading control.
(A) A plasmid system (inset) is used to control the expression of the non-required gene lacZ using the strain NQ1389. lacZ expression was induced using cTc to varying degrees at the moment of glucose depletion (time = 0 h) using the indicated range of cTc concentrations. Diauxic growth conditions with glucose and acetate, same as in Fig. 1.
(a,b) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL-1β (2 ng/ml) (a) or TNF (2 ng/ml) (b) for 24 h, then stimulated with insulin (200 nM) for 10 min. p-Akt (Ser473) was determined by flow cytometry (right) with quantification shown in the graph at left (a). p-Akt (Ser473) was determined by immunoblotting (b).
C Co-IP assay of OST1 with EGR2 in vivo. 35S:HF-OST1/Super:EGR2-Myc or 35S:HF-OST1/Super:Myc was expressed in N. benthamiana leaves. Total proteins were immunoprecipitated with anti-Myc agarose beads, and the co-immunoprecipitation products were subjected to immunoblot analysis. EGR2-Myc and HF-OST1 were detected with anti-Myc and anti-HA antibodies, respectively.
Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. Cell of patient A transfected with mRFP-GFP-LC3, labeled with LysoTracker, and imaged live by confocal microscopy under serum-starved conditions. Insert shows red+blue autolysosomes (arrowheads) and a green+red autophagosome devoid of LysoTracker signal (arrow). Scale bars 10 μm or 2 μm (inserts).
Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). Frequency of IL-22+ γδ T cells following culture without (−) or with 40ng/ml recombinant IL-23
(C) Average network growth rates for indicated WAVE1 variants or mCherry as a negative control (N= 57-73 beads per condition). Errors bars represent SD.
B. Barbed end depolymerization rate as a function of the exposure time to either 100 nM nGr-Tpm1.8 alone ("no MICAL1"), or supplemented with 100 nM MICAL1 and 12 µM NADPH. N = 31 (1 experiment) and 29 (1 experiment) filaments, without and with MICAL1, respectively. Lines: mean velocity. Shaded surfaces: S.D.
HeLa CCL2 cells stably expressing control vector only (+vector), β-catenin WT (+WTR), S60A mutant (+S60AR), or S60D mutant (+S60DR) were generated using lentiviral infection and then infected with β-catenin knockdown lentivirus (shβ-catenin). Band intensities were quantified using the ImageJ program and normalized with α-Tubulin, and the relative values of band intensities are marked. Cytokinesis-defective cellular phenotypes of each stable cell line were observed under a microscope using immunostained samples (B)
D) The effect of different amounts of EBV-LMP1 on the binding of ANT1-VDAC1 was detected by CO-IP. Data are presented as means ± S.E.M. (paired t-test, n = 3, biological replicates per group, * p < 0.05, ** p < 0.01).
B. Crossovers per 200 kb per F2 plotted along the Arabidopsis chromosomes. Mean values are shown by horizontal dashed lines. SNPs per 200 kb are plotted and shaded in colour (Col×Ws (purple), Col×Bur (Lawrence et al, 2019) (dark green), Col×Ct (light blue), Col×Ler F2 populations with 17,077 (red) and 1,840 (blue) crossovers (Serra et al, 2018b; Rowan et al, 2019) and Col×CLC (pink/light green, where pink indicates Cvi SNPs and green indicates Ler SNPs)). The positions of telomeres (TEL) and centromeres (CEN) are indicated. The number of crossovers analysed is printed inset. C. Data as for B, but analyzing crossovers (lines) or SNPs (shading) along proportionally scaled chromosome arms, orientated from telomere (TEL) to centromere (CEN).
The model predicts the competitive outcome will depend on the resource supply fractions with exclusion occurring as the supply approaches entirely alanine or entirely glutamate.
N. Cell cycle analysis via PI staining followed by flow cytometry for the cells with Pbk overexpression. PIME cells streadily transduced with vector, Pbk-WT, and Pbk-Mut expression plasmids.
44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. C Quantification of cellular and mitochondrial ROS using CellROX/ MitoSOX, indicated by MFI FITC/ MFI PE in human Pan/CD4+/CD8+ T cells, n=9/10/10 (CellROX), 12/11/12 (MitoSOX) individual human subjects. Data information: Data depicted as mean ± SEM (OCR) or as box plots with median, twenty-fifth and seventy-fifth percentiles and range Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.
G: Asynchronous (AS) FAM83DGFP/GFP/ mCherry/mCherryCSNK1A1 knockin U2OS cells were fixed and imaged. Representative images from the indicated cell cycle stages are included. Scale bars, 10 µm.
(A) Pearson correlation between ZEB1 and MITF mRNA expression levels in 467 melanomatumors from TCGA data set.
Retinal inflammatory scores of mice immunized s.c. with PBS + DMSO (Veh), IRBP + DMSO (IRBP) or IRBP + RA + IL-2 21 and 7 days before induction of EAU by s.c. injection of IRBP emulsified in CFA. Bars are mean + SEM (n=6), *p<0.05 and ***p<0.001, IRBP + IL-2 + RA vs Veh, ###p<0.001 IRBP + IL-2 + RA vs IRBP by two-way ANOVA with Tukey post hoc test.