EMBO/soda-nel huggingface dataset:
Arabidopsis HEAT SHOCK FACTOR BINDING PROTEIN is required to limit meiotic crossovers and HEI10 transcription (2022)
Figure 1

Figure 1. The hcr2 mutant is a weak hsbp allele.

Representative images of seed fluorescence segregation in 420/++ in wild type (Col) and hcr2. Scatterplots to the right show red (dsRed) and green (eGFP) fluorescence values in 420/++ Col (top) and hcr2 (bottom). Scale bars: 2 mm.

420 crossover frequencies (cM) in Col, hcr1, hcr2, hcr3, and hcr4 mutants. n ≥ 7 plants of biological replicates.

As in B, 420 crossover frequencies (cM) in Col, hcr2/HCR2, hcr2/hcr2, and individual hcr2 BC1F2 plants. n ≥ 6 plants of biological replicates.

Schematic diagram of the HSBP locus and position of the hcr2 (hsbp-3) substitution (red asterisk). Black boxes, exons; gray boxes, UTRs; introns, black lines. The conserved splicing sequence of AT-AC class introns is underlined. Primer positions for the RT-PCR and RT-qPCR analyses are indicated by arrows.

End-point RT-PCR analysis of HSBP in Col, hsbp-3, hsbp-2 and hsbp-1. Hash and asterisk indicate aberrant long and short splicing variants of HSBP in hsbp-3, respectively. Image J was used to measure relative PCR band intensity for hsbp-3 (53%), hsbp-2 (9%) and hsbp-1 (70%). TUB2 was used as an internal control.

As in E, but showing immunoblot analysis of HSBP. hsbp-3, hsbp-2 and hsbp-1 accumulate about 58%, 17% and 77% of HSBP levels, respectively. Coomassie-stained membrane was used as a loading control.

As in B, 420 crossover frequencies in Col, hsbp-3, and hsbp-3 T1 lines harboring the HSBP or HSBP-myc transgene. n ≥ 6 plants of biological replicates.

As in B, 420 crossover frequencies (cM) in Col, hsbp-3, and meiMIGS-HSBP T1 transgenic plants. n ≥ 6 plants of biological replicates.

As in B, but showing I3bc crossover frequency (cM) in Col, hsbp-3, hsbp-3/hsbp-2 F1 hybrid, and hspb-2 plants. n ≥ 5 plants of biological replicates.

Data information: (E, F) Experiments were performed at least three times. (B, G, H) Red dots and horizontal lines indicate mean ± s.d. of cM values from individual plants (one-sided Welch's t-test). Black dots represent cM values of individual plants. (I) Colored dots represent cM values from individual plants.

Source data are available online for this figure.
Arabidopsis HEAT SHOCK FACTOR BINDING PROTEIN is required to limit meiotic crossovers and HEI10 transcription (2022)
Figure 6

Figure 6. Increased MlH1 and HEI10 foci in hsbp-3.

Representative images of meiocyte spreads stained with DAPI in Col and hsbp-3 at the indicated meiotic stages. Scale bars: 10 μm.

B-D Silique lengths (B), seed numbers (C), and pollen viability (D) in Col, hsbp-3, and hsbp-2. Red dots and horizontal lines indicate mean ± s.d. of cM values (one-sided Welch's t-test). n = 30 siliques of biological replicates (B, C). n = 10 plants of biological replicates (D).

Representative images of ASY1 (green) and RAD51 (red) immunostaining in Col and hsbp-3. Nuclei spreads were stained with DAPI (blue). Scale bars: 10 μm.

Quantification of RAD51 foci numbers per cell in Col (blue) and hsbp-3 (red). n = 20 cells of biological replicates.

Representative images of MLH1 (red) immunostaining in Col, hsbp-3, hsbp-2, Col/Ler, and meiMIGS-HSBP Col/Ler. Nuclear DNA was stained with DAPI. Scale bar: 5 μm.

Quantification of the number of MLH1 foci per cell shown in (G). n ≥ 32 cells of biological replicates.

Representative images of HEI10 (red) and ASY1 (green) immunostaining in Col and hsbp-3. Scale bar: 2 μm.

Quantification of the number of HEI10 foci per cell shown in (I). n ≥ 21 cells of biological replicates.

Immunoblot analysis of HEI10 and HEI10-Myc in Col, hsbp-3, HEI10-myc and HEI10-myc plants. Experiments were performed at least three times.

Representative images of HSBP (green) immunostaining during meiosis. Nuclei spreads were stained with DAPI. Scale bars: 5 μm.
Data information: (F, H, J) Black dots and horizontal lines indicate mean ± s.d. of values (Wilcoxon test).

Source data are available online for this figure.

Expanded View figure legends
Clock component OsPRR73 positively regulates rice salt tolerance by modulating OsHKT2;1-mediated sodium homeostasis (2020)
Figure 1

Fig 1. OsPRR73 positively regulates salinity tolerance in rice.

A Schematic diagram showing the T-DNA insertion sites in rice osprr73T-DNA mutant (hereafter named as osprr73).

B OsPRR73 confers salinity stress tolerance in rice. The seedlings of Dongjin (DJ, the wild type control), osprr73, and the complementation line of osprr73 (abbreviated as Com-L1) grown under 12 h light/12 h dark conditions for 28 days (left panel), transferred to 180 mM NaCl for 21 days (middle panel) and recovered for 12 and 24 days (the two right panels) respectively. Scale bar, 5 cm.

C Statistical analysis of the survival rate of DJ, osprr73 and Com-L1 plants in (B) after recovery 12 days. Data are presented as mean ± SD. (n from 4 biological replicates, and 24 plants were tested in each of biological replicates). (***) P ≤ 0.001 were generated by student's t-test.

D Diagram showing the target and mutated sites of OsPPR73 by CRISPR/Cas9-based genome editing. The PR and CCT are the abbreviations of Pseudo-Response and COSTANS, COSTANS-LIKE and TOC1 domains respectively. The target sequences within the first exon of OsPRR73 were shown with the green boxes. PAM sequences were labelled with underline within the green boxes.

E Null mutant of osprr73 in Nipponbare (NIP) background by genome editing displayed hypersensitivity to NaCl treatment. Scale bar, 5 cm.

F Survival rate of NIP (wild type of CRISPR lines) and osprr73-C (C stands for CRISPR) plants in (E) after recovery 9 days. Data are presented as mean ± SD. n = 4 biological replicates, 24 plants for each biological replicate. (*) P ≤0.05 and (***) P ≤ 0.001 were generated by student's t-test.

G-H Quantiﬁcation of chlorophyll content and electrolyte leakage of WT and osprr73 mutants with or without 180 mM NaCl treatment for 7 days. Data are presented as mean ± SD. n = 3, biological replicates, and asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01, (***) P ≤ 0.001.
Clock component OsPRR73 positively regulates rice salt tolerance by modulating OsHKT2;1-mediated sodium homeostasis (2020)
Figure 3

Fig 3. The osprr73 mutant displays specific hypersensitivity to sodium ion stress.

A, B The Na+ and K+ contents in the shoots and roots of DJ and osprr73 mutant plants with or without 180 mM NaCl treatment for 7 days. Na+ (A) and K+ (B) contents were measured with ICP method. Data represent means ± SD, (n = 3, biological replicates). (**) P ≤ 0.001 and (*) P ≤ 0.001 indicate significant difference by student t-test. The abbreviation of n.s. stands for no significant.

C Ratio of Na+ to K+ was calculated with their respective content in (A) and (B). Data represent mean ± SD (n = 3, biological replicates). For each of biological replicates, 6 individual plants were measured. The asterisk represents significant difference among means by student's t-test with (*) P ≤0.05, and n.s. indicates no significant.

D Four-weeks old seedlings of DJ and osprr73 were treated with 90 mM Na2SO4 for 14 d (middle panel) and recovered for additional 7 days (right panel). Scale bar, 5 cm.

E Statistical analysis of survival rates with Na2SO4 treatment in (D). Data represent means ± SD. n = 3 biological replicates, 24 plants for each biological replicate. (***) P ≤ 0.001 indicates significant difference by student t-test.

F The seedlings of DJ and osprr73 mutant were treated with 90 mM MgCl2 for 14 days (middle panel) and recovered for 7 days (right panel). Scale bar, 5 cm.

G Statistical analysis of survival rates with MgCl2 treatment in (F). The survival rate of DJ and osprr73 plants in MgCl2 stress were calculated after recovery 7 days. Data represent means ± SD. n = 2 biological replicates, 24 plants of each replicate.

H The seedlings of DJ and osprr73 mutant were treated with 180 mM mannitol for 36 days (middle panel) and recovered for 7 days (right panel). Scale bar, 5 cm.

I The survival rate of DJ and osprr73 plants in mannitol stress. Data represent means ± SD. n = 3 biological replicates, 24 plants of each replicate. The abbreviation of n.s. stands for no significant generated by student t-test.
Clock component OsPRR73 positively regulates rice salt tolerance by modulating OsHKT2;1-mediated sodium homeostasis (2020)
Figure 4

Fig 4. OsPRR73 binds OsHKT2;1 promoter to repress its transcription.

A, B The expression level of OsHKT2;1 and OsHKT1;5 in osprr73 mutant and the wild control Dongjin (DJ) plants with or without salinity stress in multiple time points. DJ Data represent means ± SD. n = 3, technical replicates. The representative data is from at least two individual biological replicates. The asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01 and (***) P ≤ 0.001.

C Transient expression assay in the infiltrated leaves of N. benthamiana showing OsPRR73 can repress OsHKT2;1 transcription. Left panel showing the bioluminescence signal of OsHKT2;1 and OsPRR73. Bioluminescence of co-transformation of either GFP or 35S: GFP-OsPRR73 (GFP-OsPRR73) with OsHKT2;1pro: LUC in N. benthamiana.

D Quantiﬁcation of bioluminescence intensity of OsHKT2;1pro: LUC shown in (C). Data represent means ± SD. n = 10. The asterisks indicate the significant difference by student's t-test with (***) P ≤ 0.001. At least two biological replicates were conducted with similar results.

E ChIP-qPCR assay showing the enriched DNA fragments including the S3 and S4 regions of OsHKT2;1 promoter by OsPRR73, compared to wild type DJ controls. The amplicon of Ubiquitin was taken as a negative control. Two-week-old seedlings were harvested at ZT12. Data represent means ± SD (n =3, technical repeats). The experiments were performed at least two biological replicates with similar result. Top scheme indicates the locations of amplicons for ChIP analysis. The asterisks represent significant difference among means by student's t-test with (**) P ≤ 0.01.

F EMSA assay with the OsPRR73 incubated with the probes designed for the S4 region of OsHKT2;1 promoter. Unlabelled probes were used as competitor with indicated folds. MBP alone was used as negative control. Arrowhead marks the shifted positive bands.
Clock component OsPRR73 positively regulates rice salt tolerance by modulating OsHKT2;1-mediated sodium homeostasis (2020)
Figure 5

Fig 5. OsPRR73 physically interacts with HDAC10 in nucleus.

A Coimmunoprecipitation assay showing the physical interaction between OsPRR73 and HDAC10 in vivo. CoIP was conducted with GFP-Trap beads, and GFP and FLAG antibodies were used to detect OsPRR73 and HDAC10 in immunoprecipitants as indicated.

B Split luciferase complementation (SLC) assay showing OsPRR73 interacts with HDAC10 in the co-infiltrated N.benthamiana leaves.

C Bimolecular-fluorescence complementation (BiFC) assay shows the interaction between OsPRR73 and HDAC10 occurs in nucleus. BF represents bright field. Scale bar, 40 µm.

D N-terminus of OsPRR73 containing PR domain is required and sufficient for interacting with HDAC10. Upper panel shows the schematic illustration of full-length and the truncated OsPRR73 proteins. CoIP was performed with GFP-Trap beads, and immunoprecipitants were detected by FLAG for HADC10 and GFP for truncated proteins of OsPRR73 respectively.

E, F Chromatin Immunoprecipitation-qPCR (ChIP-qPCR) assay to detect the enriched promoter regions of OsHKT2;1 by using the H3K9ac (E) and H3 (F) antibodies. Two-week-old seedlings were harvested at ZT12. The locations of amplicons were described in Figure 4E. Data represent means ± SD. n = 3 technical replicates. The asterisk indicates the significant difference by student's t-test with (*) P ≤ 0.05. The experiments were repeated at least twice with similar result.
Clock component OsPRR73 positively regulates rice salt tolerance by modulating OsHKT2;1-mediated sodium homeostasis (2020)
Figure 6

Fig 6. OsHKT2;1 negatively regulates salinity tolerance in rice.

A Nucleotide sequence showing the mutated site of OsHKT2;1 generated via CRISPR/Cas9-based genome editing. Green box indicates the CRISPR target sequence, which locates on the first exon of OsHKT2;1. PAM sequences were labelled with underline within the green boxes.

B Sanger sequencing confirmed the oshkt2;1 mutant harbor a 1-bp insertion (pointed by red arrowheads).

C Alignment the amino acid sequences of OsHKT2;1, and its mutated version showing a premature stop codon occurred in the ORF of OsHKT2;1.

D The oshkt2;1 seedling is more tolerant to the treatment of NaCl. The oshkt2;1 and NIP, its wild type, four-week-old seedlings grown under NaCl conditions for 0 days (left panel), then were transferred to 200 mM NaCl for 28 days (middle panel), and recovered for 7 days (right panel). Scale bar, 5 cm.

E Statistical analysis of survival rate in (D). Data represent means ± SD. n = 3 biological replicates, 24 plants of each replicate. (*) P ≤ 0.05 was generated by student's t-test.
Clock component OsPRR73 positively regulates rice salt tolerance by modulating OsHKT2;1-mediated sodium homeostasis (2020)
Figure 7

Fig 7. ROS was highly accumulation in osprr73 mutant under salinity stress.

A Malondialdehyde (MDA) content in leaves of 14-d-old DJ and osprr73 plants treated by 180 mM NaCl for 3 d and normal condition. Data represent means ± SD. n = 6, biological replicates. The asterisks indicate the significant difference (***) P ≤ 0.001 by student's t-test.

B DAB staining showing the greater ROS accumulation in the leaves of osprr73 mutant treated by 180 mM NaCl treatment for 3 days. Scale bar, 1 cm.

C H2O2 concentration was detected by using H2DCFDA in the root tip of DJ and osprr73 mutant plants. Scale bar, 5 mm. -NaCl and +NaCl represented the untreated or treated plants by NaCl respectively.

D Quantitative analysis of H2O2 concentration in the root of DJ and osprr73 mutant. Data represent means ± SD. n ≥ 6. The asterisks indicate the significant difference (***) P ≤ 0.001 by student's t-test.

E, F Measurement of SOD (Superoxide dismutase) (E) and CAT (Catalase) (F) enzymes activity in shoots of DJ and osprr73 seedlings with or without NaCl stress. Data represent means ± SD. n = 3 technical replicates. Data are representative from two independent biological replicates with similar result. The asterisks indicate the significant difference (*) P ≤ 0.05 by student's t-test.
Activation loop phosphorylation regulates B-Raf in vivo and transformation by BRAF mutants (2015)
Figure 1

Figure 1. Loss of the phospho-acceptor sites of the TVKS-motif impairs the signaling potential of wildtype B-Raf and some of its gain-of-function mutants. (A) The TVKS/T-motif has been conserved throughout metazoan evolution. The phospho-acceptor sites are indicated in red, the DFG- and APE motif flanking the activation loop (AL) are indicated in blue. Residues differing from human B-Raf are shown in grey or in red/blue with reduced intensity. (B) B-RafE586K signals independently from Ras-GTP, but requires an intact AL. The MEK-ERK activation potential of the indicated HA-tagged B-Raf mutants was assessed by Western blotting using TCLs from transiently transfected Plat-E cells. (C) B-RafCAAX but not the B-RafV600E oncoprotein signals independently from Ras-GTP, but requires an intact AL. Same experimental set-up as in (B). (D) Quantification of experiments shown in (B) and (C). The signal elicited by the individual reference proteins (B-Rafwt, B-RafE586K, B-RafV600E and B-RafCAAX) was set in each analysis to 100%. n = 3, mean + SEM, t-test, * p < 0.05, ** p < 0.01
Activation loop phosphorylation regulates B-Raf in vivo and transformation by BRAF mutants (2015)
Figure 2

Figure 2. Relase of oncogenic H-Ras leads to hyperphosphorylation of MEK by kinase dead B-RAF mutants, but not by B-RafAVKA. (A) Braf-/-;ERTmHRasG12V MEFs were reconstituted with HA-tagged hBRAF constructs or empty vector (EV). Infected cells were either treated with 4-HT (1µM) to induce oncogenic Ras activation or with ethanol (OH) as a control. (B) Quantifications of pMEK (left) and pERK (right) normed to WT level. n = 3, mean + SEM, t-test, ** p < 0.01. (C) The AVKA mutation has no discernible influence on Ras-induced paradoxical ERK activation triggered by the D594A mutation. Experimental set-up as in (A), shown are lysates from 4-HT treated MEFs. (D) Quantification of pMEK and pERK levels normed to HA expression. n = 3, mean + SEM, t-test, ** p < 0.01. (E) Top: Braf-/-;ERTmHRasG12V MEFs were reconstituted with the indicated B-Raf constructs. Following induction of Ras signaling, HA-B-Raf proteins were purified using anti-HA antibodies and subject to Western blotting to analyze immunoprecipitation of Raf-1. Bottom: Quantification of co-immunoprecipitated Raf-1 normed to HA-B-Raf levels. n = 3, mean + SEM, t-test, * p < 0.05.
Activation loop phosphorylation regulates B-Raf in vivo and transformation by BRAF mutants (2015)
Figure 3

Figure 3. Generation of the conditional BrafAVKA knock-in mouse. (A) Targeting strategy. Top: Targeting vector pSC-floxAVKA and a part of the murine Braf locus containing exons E14–E16 (not drawn to scale). Middle: Braf locus after homologous recombination with the targeting vector replacing E15 with a loxP-flanked (floxed) minigene (MG; E15-20) segment. pA = polyadenylation signals; SA = splice acceptor. LoxP and Frt sites are indicated by red triangles and blue hexagons, respectively. Bottom: The modified Braf locus after Flp-e and Cre mediated recombination. (B) Genomic PCR with the primers indicated as red arrows in (A) showing 3 out of 9 clones being positive for homologous recombination. (C) Southern blot analysis of genomic DNA of parental W4 ES cells, wildtype MEFs and the ES cell clones #286 and #273 (with recombined B-raffloxAVKA allele). Genomic DNA was digested by AseI and analyzed using the external probe indicated in (A). (D) Genomic PCR showing Cre-mediated recombination of the BraffloxAVKA allele in vivo using the primer pair E14fwd and E15rev (indicated as blue arrows in A). (E) Electropherograms of sequenced RT-PCR amplicons generated using splenic RNA of a Braf+/floxAVKA (left) and a Braf +/AVKA;Cre mouse (right). RT-PCR amplicons were generated using the primers E14fwd and E16revNEW. Note the double peaks at the first position corresponding to the threonine/serine and alanine codons (indicated by red arrows).
Activation loop phosphorylation regulates B-Raf in vivo and transformation by BRAF mutants (2015)
Figure 4

Figure 4. Basic comparison and immune phenotyping between homozygous Braf WT and AVKA mice. (A) Weight at weaning of BrafAVKA mice from two different founders showed no significant differences. 215: n = WT = 24/ Heterozygous = 32/ AVKA = 19; 273: n = WT = 114/ Heterozygous = 117/ AVKA = 52, mean + SEM, t-test (B) Homozygous BrafAVKA mice show reduced splenic cellularity. n = 7, mean + SEM, t-test, * p < 0.05. (C-E) Relative number of CD8+ T cells. FACS analysis shows an increase in naïve cells (C), whereas differentiated TEM (D) and Tregs (E) reveal a reduction in BrafAVKA mice compared to WT. n = 8, mean + SEM, t-test, * p < 0.05, ** p < 0.01. (F) Primary B cells of BrafAVKA mice show reduced CD69 surface expression upon LPS and anti-IgM stimulation. n = 3, mean + SEM, test, ** p < 0.01. (G) Primary B cells also show reduced proliferation upon stimulation compared with WT B cells. n = 3, mean + SEM, test, ** p < 0.01
Activation loop phosphorylation regulates B-Raf in vivo and transformation by BRAF mutants (2015)
Figure 5

Figure 5. MEK and ERK activity is drastically impaired in the brain of AVKA knock-in mice. (A) Facial appearance of BrafAVKA mice. (B) Top: In vitro kinase (IVK) assay of B-Raf complexes purified from either WT or BrafAVKA knock-in brains from both founder strains revealed a reduced MEK phosphorylation potential regardless of their purification from NLB or RIPA lysates. Botttom: Quantification of IVK differentials: #215 samples indicated in black, #273 samples in grey. (C) Western blot analysis showing reduced pMEK and pERK levels in BrafAVKA brains. n = 5, mean + SEM, t-test, ** < 0.01. (D) Immunohistochemistry showing reduced pERK levels in Purkinje cells of homozygous BrafAVKA mice. Scale bar indicates 10x magnification.
Activation loop phosphorylation regulates B-Raf in vivo and transformation by BRAF mutants (2015)
Figure 6

Figure 6. Characterization of the homo- and hetero-dimerization potential of B-RafAVKA. (A) Total brain lysates of WT and BrafAVKA mice were subject to immunoprecipitation (IP) using either anti-B-Raf antibodies or non-specific IgG as negative control. The co-purified proteins were visualized using the indicated antibodies and quantified. Ksr1 is significantly decreased in B-RafAVKA complexes while the other proteins are equally bound to B-RafWT or B-RafAVKA. n = 3, mean + SEM, t-test, * p < 0.05. (B) Anti-Myc IP of Plat-E cells transfected with Myc-tagged Raf-1 and different HA-B-Raf constructs. Western blot analysis showed that B-RafAVKA is not impaired in its ability to interact with Raf-1. (C) Anti-Myc IP of Plat-E cells expressing different Myc- and HA-B-Raf proteins. Note that B-RafAVKA is not impaired in its ability to interact with other B-Raf proteins, while homo-dimerization of B-RafEVKD is enhanced. (D) Top: Exemplary Western blot analysis of EGF stimulated primary astrocytes. Mutant astrocytes show drastic reduction in basic and stimulated levels of pMEK and pERK. Bottom: Quantification of all Western blot samples. WT: n = 7, AVKA: n= 6, mean + SEM, t-test, * p <0.05, ** p < 0.01.
Activation loop phosphorylation regulates B-Raf in vivo and transformation by BRAF mutants (2015)
Figure 7

Figure 7. Phenotypic characterization of conditional BrafAVKA MEFs. (A) MEFs expressing B-RafWT or B-RafAVKA were stimulated with EGF for the indicated time points (min) and analyzed by Western blotting. (B) IP of EGF stimulated MEFs expressing B-RafWT or B-RafAVKA. Sorafenib was used to increase complex build up. (C) Proliferation of two different MEF pools expressing either B-RafWT or B-RafAVKA. MEFs expressing B-RafAVKA show a strong reduction in growth rate. n = 3, mean + SEM, t-test, ** p < 0.01, *** p < 0.001. (D) Cell morphology of B-RafAVKA expressing MEFs are enlarged and present with abnormal nuclei (E) MEFs expressing B-RafAVKA show a significant increase in caspase-3 activity. n = 3, mean + SEM, t-test, ** p < 0.01. (F) Decay of TAg expression in MEFs following dox withdrawal. (G) Morphology of MEFs expressing B-Rafwt or B-RafAVKA in the presence or absence of TAg.
Activation loop phosphorylation regulates B-Raf in vivo and transformation by BRAF mutants (2015)
Figure 8

Figure 8. The AVKA mutation is able to block transformation potential of B-RAF mutants and model of B-Raf activation incorporating the roles of the activation segment and the DIF. (A) MEFs were then infected with retroviral vectors encoding the indicated B-Raf proteins. Giemsa staining revealed transformed and hyper-proliferated cells by dark purple colour. (B) Quantification of Focus formation potential of the B-Raf mutants used in (A). n = 3, mean + SEM, t-test, *** p < 0.001. (C) Following recruitment by Ras-GTP, B-Raf forms homo- or heterodimers with the assistance of 14-3-3 proteins. This loosely dimerized and membrane-bound state is imitated by the B-RafE586K and B-RafCAAX mutants, which still require an intact DIF and AL phosphorylation. Within the dimer, one protomer becomes phosphorylated at its TVKS-motif in cis (depicted by black bent arrow) leading to re-orientation of the DIF and induction of a conformational change in the other protomer (blue bent arrow) followed by phosphorylation of the AS in the other protomer in cis. B-RafV600E is locked in the highly active state and thus signals independent of an intact TVKS-motif or DIF. See text for further details.