Anti-RIP3 (phospho S232) antibody [EPR9516(N)-25]

• WB

Supplier Data

Western blot - Anti-RIP3 (phospho S232) antibody [EPR9516(N)-25] (AB195117)

Blocking/Dilution buffer : 5% NFDM/TBST.

Lysates were provided by Xiaodong Wang, National Institute of Biological Sciences, Beijing

All lanes:

Western blot - Anti-RIP3 (phospho S232) antibody [EPR9516(N)-25] (ab195117) at 1/2000 dilution

Lane 1:

Untreated TSE (WT) whole cell lysate at 10 µg

Lane 2:

TSE (WT) whole cell lysate treated with 20 ng/ml TNF alpha,100 nM Smac mimetic, and 20 µM z-VAD for 12 h and then harvested at 10 µg

Lane 3:

Untreated TSE (KO RIP3) whole cell lysate at 10 µg

Lane 4:

TSE (KO RIP3) whole cell lysate treated with 20 ng/ml TNF alpha,100 nM Smac mimetic, and 20 µM z-VAD for 12 h and then harvested at 10 µg

Lane 5:

Untreated TSE (KO MLKL) whole cell lysate at 10 µg

Lane 6:

TSE (KO MLKL) whole cell lysate treated with 20 ng/ml TNF alpha,100 nM Smac mimetic, and 20 µM z-VAD for 12 h and then harvested 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 1/1000 dilution

Predicted band size: 57 kDa

Observed band size: 53 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-RIP3 (phospho S232) antibody [EPR9516(N)-25] (AB195117)

Blocking and diluting buffer : 5% NFDM/TBST.

ab181602 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 S232) antibody [EPR9516(N)-25] (ab195117) 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

false

Exposure time: 180s

• WB

Unknown

Western blot - Anti-RIP3 (phospho S232) antibody [EPR9516(N)-25] (AB195117)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-RIP3 (phospho S232) antibody [EPR9516(N)-25] (ab195117) at 1/10000 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 phosphatase 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 1/100000 dilution

Predicted band size: 57 kDa

Observed band size: 53 kDa

false

Exposure time: 3min

• ELISA

Supplier Data

ELISA - Anti-RIP3 (phospho S232) antibody [EPR9516(N)-25] (AB195117)

Serially diluted ab195117 was bound to immobilized phospho or non-phospho (control) peptide demonstrating minimal cross-reactivity to the non-phosphorylated residue. Antigen - RIP3 (phospho S232) phospho peptide;RIP3 non-phospho peptide at 250 ng/ml. Primary antibody concentration range : 0 - 500 ng/ml. Secondary antibody - Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) at 1 : 2500.

• Dot

Supplier Data

Dot Blot - Anti-RIP3 (phospho S232) antibody [EPR9516(N)-25] (AB195117)

Primary antibody : ab195117 at 1 : 1000 dilution (1.6μg/ml), secondary antibody : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 1000 dilution, Blocking and dilution buffer : 5% NFDM/TBST, Lane 1 : RIP3 (phospho S232) phospho peptide, Lane 2 : RIP3 Non-phospho peptide, Exposure time : 3 minutes.

We observed weak binding to the non-phospho peptide at very high concentrations.

• WB

CiteAb

Western blot - Anti-RIP3 (phospho S232) antibody [EPR9516(N)-25] (AB195117)

RIP3 (phospho S232) western blot using anti-RIP3 (phospho S232) antibody [EPR9516(N)-25] ab195117. Publication image and figure legend from Siegmund, D., Ehrenschwender, M., et al., 2018, Cell Death Dis, PubMed 30206205.

ab195117 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 ab195117 please see the product overview.

TNFR2-induced upregulation of A20 and TRAF1 is not an epiphenomenon of TNFR2-triggered necroptosis.a Wild-type, RIPK3-, and MLKL-knockout macrophages were stimulated as indicated with human TNF (100 ng/ml), TNC-sc(mu)TNF80 (200 ng/ml), and ZVAD (20 μM). Cell viability was quantified after 36 h using MTT. Data points of five independent experiments were shown with mean ± SEM (***p < 0.001). b, c RIPK3- (b) and MLKL- (c) knockout macrophages along with wild-type macrophages were treated overnight with the indicated combinations of 200 ng/ml TNC-sc(mu)TNF80, 100 ng/ml human TNF, 20 μM ZVAD, and necrostatin-1 (45 μM). Total cell lysates were prepared and analyzed by western blot. d Wild-type and TNFR2-knockout macrophages were stimulated for 7 h with human TNF (100 ng/ml) or TNC-sc(mu)TNF80 (200 ng/ml) in the presence and absence of ZVAD (20 μM), and total cell lysates were analyzed by western blot for RIPK1 phosphorylation. e The various macrophage types were stimulated for 36 h with the indicated mixtures of TNC-sc(mu)TNF80 (200 ng/ml), ZVAD (20 μM), and necrostatin-1 (45 μM). IL-1β in the supernatants was determined by ELISA assay. Shown are the mean ± SEM of five or six independent experiments. ***p < 0.001

false