sds–page and scanning densitometry  (Molecular Dynamics Inc)

Journal:

Article Title: A Cell Signal Pathway Involving Laminin-5, ?3?1 Integrin, and Mitogen-activated Protein Kinase Can Regulate Epithelial Cell Proliferation

doi:

Figure Lengend Snippet: LN5-rich matrices of either 804G cells (lane 1) or MCF-10A cells (lane 2) prepared according to Langhofer et al. (1993) as well as recombinant human α3 chain G domain (lanes 3 and 4) were processed for SDS-PAGE on either 6% (lanes 1 and 2) or 7.5% (lanes 3 and 4) gels. The separated proteins were then transferred to nitrocellulose and immunoblotted with CM6 (lane 1), RG13 (lane 2 and 3), and control IgG (lane 4) antibodies. CM6 antibodies recognize rat 160-kDa α3 chain (lane 1) (Baker et al., 1996 ), whereas RG13 recognizes human 160-kDa α3 chain (lane 2). Whereas RG13 shows reactivity with the G domain of human α3 chain in lane 3, the control IgG does not (lane 4). Molecular mass standards of 194, 120, 87, 64, 52, 39, and 26 kDa are indicated (from top to bottom).

Article Snippet: SDS-PAGE, Western Immunoblots, and Scanning Densitometry SDS-PAGE and immunoblotting were carried out as described previously with the exception that blots were developed using a chemiluminescence kit (Pierce) ( Zackroff et al. , 1984 ; Klatte et al. , 1989 ).

Techniques: Recombinant, SDS Page

Journal:

Article Title: A Cell Signal Pathway Involving Laminin-5, ?3?1 Integrin, and Mitogen-activated Protein Kinase Can Regulate Epithelial Cell Proliferation

doi:

Figure Lengend Snippet: (A) MAPK assay blot in which 804G cells were plated onto tissue culture plastic or surfaces coated with 50 μg/ml RTC, 25 μg/ml FN, 25 μg/ml LN1, or 1 μg/ml hLN5 for 48 hr. As indicated, the 804G cells were maintained in medium supplemented with either 50 μg/ml IgG control antibody or 50 μg/ml CM6 antibodies. After 48 hr the cells were scraped into gel sample buffer, processed for SDS-PAGE, transferred to nitrocellulose, and immunoblotted with either anti-ACTIVE MAPK p42/p44 to determine phosphorylated p42/p44 (lower panel) or a probe for total p42/p44 (upper panel). (B–D) Scan analyses of MAPK blots of 804G cells (B) and MCF-10A (C and D) were undertaken using the Bio-Rad Molecular Analyst program. The “amount” of total p42/p44 in each sample was normalized to that observed in IgG control-treated specimens, and then the levels of activated p42/p44 were appropriately adjusted. We then calculated the % phosphorylated p42/p44 for each specimen relative to that observed in the IgG control samples. The culture conditions and concentrations of antibodies used are identical to those shown in Figures ​Figures4,4, ​,6A,6A, and ​and88.

Article Snippet: SDS-PAGE, Western Immunoblots, and Scanning Densitometry SDS-PAGE and immunoblotting were carried out as described previously with the exception that blots were developed using a chemiluminescence kit (Pierce) ( Zackroff et al. , 1984 ; Klatte et al. , 1989 ).

Techniques: SDS Page