- PA1-39503 - Invitrogen Antibodies RPS6 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of U-2 OS (Lane 1), U-2 OS treated with IGF (50 ng/mL for 6 hr) (Lane 2), U-2 OS treated with Rapamycin (50 nM for 4 hr) followed by treatment with IGF (50 ng/mL for 6hr) (Lane 3), A-431 (Lane 4), A431 treated with EGF (200 ng/mL for 10 min) and A-431 treated with Rapamycin (50 nM for 4 hr) followed by EGF treatment (200 ng/mL for 10 min). The blot was probed with Anti- Phospho-S6 (Ser240, Ser244) Monoclonal Antibody (Product # PA1-39503, 1:500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 32 kDa band corresponding to Phospho-S6 (Ser235, Ser236) was enhanced upon IGF and EGF treatment and was decreased upon rapamycin treatment on the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Phospho-S6 pSer240/244 (green) in HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed without (left panel) or with (right panel) a Phospho-S6 pSer240/244 polyclonal antibody (Product # PA1-39503) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:400 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin (Product # 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan or a ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Phospho-S6 (Ser240,Ser244) was performed using 70% confluent log phase U-2 OS cells treated with 50 ng/mL IGF-1 for 6hrs. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-S6 (Ser240,Ser244) Polyclonal Antibody (Product # PA1-39503) at 1:100 dilution in 0.1% BSA, incubated overnight at 4 degree Celsius and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents cells treated with antagonist, Rapamycin (50 nM for 4hrs) followed by IGF (50 ng/mL for 6hrs), showing reduced expression of phospho-S6. Panel f shows the untreated cells showing basal expression of phospho S6. Panel g shows control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of Phospho-S6 Ribosomal pSer240/244 using anti-Phospho-S6 Ribosomal pSer240/244 Polyclonal Antibody (Product # PA5-32581) in Breast Carcinoma Cancer Tissue. The recommened dilution for this antibody in immunohistochemistry applications is 1:50.

PA1-39503