1. The adaptor protein Lad associates with the G protein β subunit and mediates chemokine-dependent T-cell migration
by Dongsu Park, Inyoung Park, Deogwon Lee, Young Bong Choi, Hyunsook Lee, and Yungdae Yun
Blood
Volume 109(12):
June 15, 2007
©2007 by American Society of Hematology

2. Lad associates with Gβ.
Lad associates with Gβ. Cos7 cells were transfected with the indicated combinations of expression plasmids encoding Lad, Gβ, and/or Gγ. After incubation for 48 hours, the cell lysates were subjected to immunoprecipitation with the antiFLAG antibody to precipitate FLAG-tagged Lad. This was followed by Western blotting with antiGβ (upper panel) or antiFLAG (lower panel) antibody.
Dongsu Park et al. Blood 2007;109:
©2007 by American Society of Hematology

3. Lad associates with Gβ in T cells upon chemokine treatment.
Lad associates with Gβ in T cells upon chemokine treatment. (A) Establishment of Jurkat T-cell clones that stably express FLAG-tagged full-length Lad (Lad) or Lad-SH2 domain (Lad-SH2). Three independent clones were selected for each transfectant, and the expression of the transfected genes was assayed by Western blotting with anti-FLAG antibody. The positive control (+) is the lysate of Cos7 cells transfected with expression plasmids encoding Lad or Lad-SH2 domain. The negative control (−) is the lysate of untransfected COS7 cells. (B) Lad associates with Gβ in T cells upon chemokine treatment. Jurkat T cells that stably express full-length Lad were activated with anti-CD3ϵ antibody (CD3), SDF-1α (S), or RANTES (R) for 10 minutes. The cell lysates were then subjected to immunoprecipitation with anti-FLAG antibody and Western blotting with anti-Gβ antibody (upper panel). The same precipitates were also analyzed by Western blotting with anti-phosphotyrosine (α-pY) antibody (middle panels) or anti-Flag antibody (lower panel). To visualize the phosphorylated Lad band more clearly, another panel from a shorter exposure of the same blot around the phosphorylated Lad band is shown below middle panel. pLAD indicates phosphorylated Lad; HC, IgG heavy chain
Dongsu Park et al. Blood 2007;109:
©2007 by American Society of Hematology

4. Lad is required for chemokine-induced T-cell migration.
Lad is required for chemokine-induced T-cell migration. (A) Suppression of endogenous Lad expression by siRNA inhibits chemokine-dependent T-cell migration. Jurkat T cell clones that stably express CCR5 were transfected with pSUPER plasmid expressing control siA or siA. Subsequently, 5 × 105 cells were loaded into the upper part of a Transwell chamber inserted into medium containing 25 ng/mL or 50 ng/mL SDF-1α or RANTES. After incubation for 2 hours, cells that had migrated into the bottom chamber were counted. The experiments were performed 3 times in duplicate, and the results were expressed as mean ± SD. Error bars represent the standard deviation. As a control, protein level of endogenous Lad was analyzed by Western blotting with antiLad antibody (inset). (B) Jurkat T-cell clones that stably express Lad (Lad) or the SH2 domain of Lad (Lad-SH2) were subjected to the chemotaxis assay described in panel A. Two independent clones were tested for each transfectant.
Dongsu Park et al. Blood 2007;109:
©2007 by American Society of Hematology

5. Lad associates with Lck and Zap-70 in response to chemokine treatment.
Lad associates with Lck and Zap-70 in response to chemokine treatment. (A) Lck associates with Lad upon chemokine treatment. Jurkat T cells that stably express Lad-FLAG (Lad) or were transfected with empty vector (Vec) were activated with anti-CD3 antibody (CD3), SDF-1α (S), or RANTES (R) for 10 minutes. The cell lysates were then subjected to immunoprecipitation with 5 μg antiFLAG antibody, and the precipitates were analyzed by immunoblotting with antiLck antibody (upper panel). The membrane was reprobed with the anti-FLAG Ab (bottom panel). (B) Zap-70 associates with Lad upon chemokine treatment. Jurkat T cells were transfected with pcDNA3.1 or the expression plasmid encoding Lad-FLAG. These cells were then stimulated for the indicated periods with SDF-1α (S) or RANTES (R), after which the cell lysates were subjected to immunoprecipitation with an anti-FLAG antibody and subsequent immunoblotting with anti–Zap-70 antibody (top panel). The membrane was reprobed with the anti-FLAG Ab (bottom panel). (C) The chemokine-dependent phosphorylation of Zap-70 is repressed by expressing hLad siRNA. Jurkat T cells were transfected with empty pSUPER or pSUPER plasmids expressing SiA or SiB and stimulated with SDF-1α (S) or RANTES (R). The cell lysates were then analyzed by immunoprecipitation with anti–Zap-70 antibody and subsequent immunoblotting with anti-phosphotyrosine antibody (4G10) (top panel). The blot was also probed with the anti–Zap-70 antibody (bottom panel). (D) Jurkat T-cell clones that stably express CCR5 were transfected with pSUPER plasmids expressing control siA or siA and stimulated with SDF-1α or RANTES. The cell lysates were analyzed by Western with anti–phosphoZap-70 (Tyr493) or anti–Zap-70 Abs.
Dongsu Park et al. Blood 2007;109:
©2007 by American Society of Hematology

6. Lad is required for the chemokine-induced activation of ERK
Lad is required for the chemokine-induced activation of ERK. (A) Jurkat T cells transfected with empty pSUPER or pSUPER plasmids expressing SiA or SiB were treated with SDF-1α (S) or RANTES (R) for 10 minutes, and the cell lysates were analyzed by Western b...
Lad is required for the chemokine-induced activation of ERK. (A) Jurkat T cells transfected with empty pSUPER or pSUPER plasmids expressing SiA or SiB were treated with SDF-1α (S) or RANTES (R) for 10 minutes, and the cell lysates were analyzed by Western blotting with anti–phospho p42/44 ERK antibody (upper panel). The same blot was reprobed with anti–p42/44 ERK antibody to control for the level of ERK (middle panel). The same lysates were also analyzed for the level of endogenous Lad by Western blotting with antiLad antibody (bottom panel). (B) Jurkat T-cell clones that stably expressed Lad or the SH2 domain of Lad were activated and processed as described in panel A.
Dongsu Park et al. Blood 2007;109:
©2007 by American Society of Hematology

7. Lad is required for the chemokine-induced activation of focal adhesion molecules such as Pyk2 and paxillin.
Lad is required for the chemokine-induced activation of focal adhesion molecules such as Pyk2 and paxillin. (A) Jurkat T-cell clones expressing CCR5 were transfected with pSUPER plasmids expressing control siA or siA and treated with SDF-1α (S) or RANTES (R) for 2 or 5 minutes. Pyk2 was immunoprecipitated and the immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine or anti-Pyk2 antibody. IgG was used as a negative control. (B) Jurkat T cells stably expressing Lad or the SH2 domain of Lad were treated with SDF-1α or RANTES and processed as described in panel A. (C) The cell lysates prepared as described in panel A were analyzed by immunoprecipitation with anti-paxillin antibody and subsequent immunoblotting with anti-phosphotyrosine antibody (4G10) (upper panel). As a protein-loading control, the blots were reprobed with anti-paxillin antibody (lower panel). (D) The same cell lysates used in panel A were analyzed for the Lad levels by Western blotting with anti-Lad antibody (upper panel) and with anti-actin antibody (lower panel).
Dongsu Park et al. Blood 2007;109:
©2007 by American Society of Hematology