Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52]

• WB

Supplier Data

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] (AB222320)

Blocking and diluting buffer concentration : 5% NFDM/TBST.

ab181602 GAPDH was used as a loading control at 1/1000000 dilution.

We recommend involving downstream protein p-MLKL as a control to validate the stimulation of p-RIP3.

Lanes 1 - 4:

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] (ab222320) at 1/1000 dilution

Lanes 5 - 8:

Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (<a href='/en-us/products/primary-antibodies/mlkl-phospho-s345-antibody-epr95152-ab196436'>ab196436</a>) at 1/1000 dilution

Lanes 1, 3, 5 and 7:

Untreated L-929 (mouse connective tissue fibroblast cell line) whole cell lysate at 20 µg

Lanes 2 and 6:

L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 8 hours whole cell lysate at 20 µg

Lanes 4 and 8:

L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 6 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 57 kDa,54 kDa

Observed band size: 53 kDa

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Exposure time: 180s

• WB

Lab

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] (AB222320)

Blocking/Dilution buffer : 5% NFDM/TBST.

Cells were lysed in 4% SDS buffer (4% SDS,10% 2-ME, 20% glycerol, 0.004% Bromophenol blue, Tris-HCl).

The images were kindly provided by our collaborator Dr. Jiahuai Han, Xiamen University.

All lanes:

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] (ab222320) at 1/500 dilution

Lane 1:

Untreated L-929 (mouse connective tissue fibroblast cell line) whole cell lysate at 20 µg

Lane 2:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate treated with 10 ng/ml TNF-a and 20 µM z-VAD for 2 hours at 20 µg

Lane 3:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate treated with 10 ng/ml TNF-a and 20 µM z-VAD for 3.5 hours at 20 µg

Lane 4:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 µM z-VAD for 6 hours at 20 µg

Lane 5:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate with RIP3 KO cells treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 µM z-VAD for 6 hours at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/5000 dilution

Predicted band size: 57 kDa

false

Exposure time: 60s

• WB

Lab

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] (AB222320)

Blocking/Dilution buffer : 5% NFDM/TBST.

The higher MW bands in Lanes 1 and 2 are extra bands.

The lysates were prepared following the 1% SDS Hot Lysate buffer method.

For Lysate preparation protocol, please refer to the protocol section of the website and/or here.

All lanes:

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] (ab222320) at 1/1000 dilution

Lane 1:

Untreated L-929 (mouse connective tissue fibroblast cell line) whole cell lysate at 10 µg

Lane 2:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 µM z-VAD for 6 hours at 10 µg

Lane 3:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 µM z-VAD for 6 hours, then treated with alkaline phosphatase for 1 hour at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 57 kDa

Observed band size: 53 kDa

false

Exposure time: 30s

• Dot

Lab

Dot Blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] (AB222320)

Dot blot analysis of RIP3 (phospho T231 + S232) labeled with ab222320 at 1/1000 dilution.

Lane 1 : RIP3 (phospho T231 + S232) peptide (aa229-239);

Lane 2 : RIP3 (phospho T231 + S232) peptide (aa223-236);

Lane 3 : RIP3 (phospho T231) peptide (aa223-239);

Lane 4 : RIP3 (phospho S232) peptide (aa223-239);

Lane 5 : RIP3 non-phospho peptide (aa223-239).

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

• WB

CiteAb

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] (AB222320)

RIP3 (phospho T231 + S232) western blot using anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] ab222320. Publication image and figure legend from Jiao, J., Wang, Y., et al., 2020, Front Pharmacol, PubMed 31998134.

ab222320 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab222320 please see the product overview.

NSA suppresses MLKL activation but not p-RIP3. Prevention of MLKL activation by treatment with 3 μM NSA for 24 h after OGD treatment. (A) NeuN and p-MLKL double staining. Cell nuclei were stained with DAPI (blue fluorescence) and neurons were stained with NeuN (red fluorescence). Pictures were taken using a fluorescence microscope (scale bar = 20 μm). (B) WB analysis of the level of p-RIP3 and p-MLKL induced by OGD, p-MLKL level was significantly reversed after NSA treatment. Data are presented as the mean ± SEM, n = 4. *p < 0.05, vs. the control group (CTL). #p < 0.05, vs. the OGD group. Data were analyzed using Student's t-test. RIP3, receptor interacting protein kinase-3; OGD, oxygen-glucose deprivation; NSA, necrosulfonamide; MLKL, mixed-lineage kinase domain-like protein; Nec-1, necrostatin-1; WB, Western blot.

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