Coupling of the TCR to Integrin Activation by SLAP-130/Fyb E. J. Peterson, M. L. Woods, S. A. Dmowski, G. Derimanov, M. S. Jordan, J. N. Wu, P. S. Myung, Q.-H. Liu, J. T. Pribila, B. D. Freedman, Y. Shimizu, G. A. Koretzky

Supplementary Material

Supplemental Figure 1. Targeting of murine SLAP-130/Fyb. (A) Schematic of the SLAP-130/Fyb locus, targeting vector, and targeted allele. (B) Southern analysis of genomic DNA. PstI digests of tail DNA were analyzed by hybridization with a probe ("A" in Supplemental fig. 1A) derived from genomic sequence 1 kb upstream of the 5´ recombination arm of the targeting vector. Probe A hybridizes with a 10.5 kb PstI fragment in the wild-type or with a 7 kb fragment in the targeted allele. Genotype interpretation is listed below the Southern blot. (C) Thymocyte lysates were probed with an antiserum raised against the carboxyl terminus of SLAP-130/Fyb (Transduction Laboratories).

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A 60 kDa species, with an intensity of less than 5% of the 130 kDa SLAP-130/Fyb band by densitometric analysis, is immunoreactive with anti-SLAP-130/Fyb antiserum in SLAP-130/Fyb+/- and -/-, but not +/+ thymocyte lysates (arrow, Supplemental fig. 1C). Northern blotting and RT-PCR (E.J. Peterson, G.A. Koretzky, unpublished observations) of SLAP-130/Fyb-/- thymic RNA detected a transcript containing non-targeted 3´ SLAP-130-/- exons fused with the neomycin resistance cDNA (in anti-sense orientation relative to SLAP-130). However, sequence analysis of the mutant mRNA revealed no potential initiation codons in frame with the SLAP-130 coding region. We investigated whether the 60 kDa species represents a functional truncated form of SLAP-130/Fyb by examining proteins associated with the SLAP-130/Fyb binder, SLP-76. No new phosphoproteins or SLAP-130/Fyb-reactive bands were detected in SLP-76 GST fusion protein pull-downs or SLP-76 immunoprecipitates from pervanadate-stimulated SLAP-130/Fyb-/- thymocyte lysates (E.J. Peterson, G.A. Koretzky, unpublished observations). Thus, if a truncated SLAP-130/Fyb protein exists in the mutant T cells, it is expressed at extremely low levels, and cannot bind SLP-76.

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Supplemental Table 1.aHematopoietic Cellularity
	bWild-type	SLAP-130/Fyb-/-	dp value
Thymocyte number (X 106); cn=4	254 +/- 88	188 +/- 66	p =0.001
Splenocyte number (X 106); n=5	94 +/- 22	82 +/- 9	NS
CD3	35 +/- 5	22 +/- 4	p =0.0001
CD4	29 +/- 5	19 +/- 2.8	p =0.002
CD8	13.5 +/- 3.1	7.8 +/- 2.2	p =0.0004
B220	52 +/- 6.8	69 +/- 1.5	p =0.001
eDX-5	3.8 +/- 1.2	2.9 +/- 0.14	NS
WBC (number/L X 103); n=7	7.8 +/- 4.1	6.9 +/- 3.3	NS
Hematocrit (%); n=5	49 +/- 8	51 +/- 5	NS
Platelets (number/L X 103); n=8	721 +/- 275	410 +/- 74	p =0.01
aMean values +/- one standard deviation are shown.
Blue font indicates "Percentage of splendocytes expressing surface antigens".
bWild-type mice were littermates or age- and strain-matched.
cThymocyte numbers were determined in 6 week old mice.
dProbability of no significant difference between mean values was calculated using a paired T test. A p value of greater than 0.05 was considered not significant (NS).
eDX-5 identifies natural killer lymphocytes.

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Supplemental Figure 2. T cell development occurs in the absence of SLAP-130/Fyb. Thymocytes or splenocytes from 8-week-old SLAP-130/Fyb+/+ or -/- mice were stained with the indicated fluorochrome-conjugated mAbs (Pharmingen). Plots represent flow cytometric analysis of live lymphocytes identified by forward/side scatter characteristics (FACscalibur). For analysis of CD25/CD44 expression, gates were set upon CD4-, CD8-, CD3- ("triple-negative") thymocytes. Quadrant numbers indicate percentages of gated cells.

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